CN116687824A - Method for preparing herba Melissae axillaris extract, and cosmetic composition - Google Patents
Method for preparing herba Melissae axillaris extract, and cosmetic composition Download PDFInfo
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- CN116687824A CN116687824A CN202310844014.0A CN202310844014A CN116687824A CN 116687824 A CN116687824 A CN 116687824A CN 202310844014 A CN202310844014 A CN 202310844014A CN 116687824 A CN116687824 A CN 116687824A
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- extract
- melissa
- alcohol
- eluent
- solution
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Classifications
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Landscapes
- Medicines Containing Plant Substances (AREA)
Abstract
The application discloses a method for preparing a melissa extract and a cosmetic composition. The method comprises the following steps: crushing, namely crushing the raw materials of the melissa officinalis to obtain crushed melissa officinalis; the extraction step comprises extracting the crushed herba Melissae axillaris with a mixed solution of alcohol and water to obtain a crude extract; filtering, namely filtering the crude extract by adopting a filter membrane to obtain filtrate; concentrating, namely vacuum concentrating the filtrate to remove partial alcohol and water to obtain crude extract; the resin purification step comprises the steps of loading the crude extract into an adsorption resin column, and performing water elution and gradient analysis of an alcohol-water solution to obtain an eluent; a decoloring step, which comprises the steps of adsorbing and decoloring eluent by using active carbon and filtering to obtain decoloring liquid; concentrating and drying, namely concentrating and drying the decolorized solution to obtain a powdery bee grass extract. The method can prepare nanometer powder herba Melissae axillaris extract, and the coumarin content is lower than detection limit.
Description
Technical Field
The application belongs to the technical field of plant extraction, and particularly relates to a method for preparing a melissa extract and a cosmetic composition.
Background
Melissa officinalis (Melissa officinalis) is perennial herb of Melissa of Labiatae, and is also called balm and Melissa officinalis. Herba Melissae axillaris can be used for treating dysentery, intestinal ulcer, swelling, arthralgia and toothache, and is also called "Happy plant".
In the related art, extracts obtained from melissa officinalis through petroleum ether, chloroform, ethyl acetate and n-butanol all show moderate to strong activities against luteal sarcoma, staphylococcus aureus and bacillus cereus. However, the extract contains components harmful to skin, and the additional impurity removal results in further reduction of active ingredients, so that the effect of the melissa extract is greatly reduced, and the application of the melissa extract is limited.
Therefore, finding a non-irritating and safe preparation method is the key point of research.
Disclosure of Invention
In view of the above, the present application provides a method for preparing a melissa extract and melissa extract, and a cosmetic composition, aiming at improving the effective ingredient in melissa extract.
In a first aspect, embodiments of the present application provide a method for preparing a melissa extract, comprising:
crushing, namely crushing the raw materials of the melissa officinalis to obtain crushed melissa officinalis;
the extraction step comprises extracting the crushed herba Melissae axillaris with a mixed solution of alcohol and water to obtain a crude extract;
filtering, namely filtering the crude extract by adopting a filter membrane to obtain filtrate;
concentrating, namely vacuum concentrating the filtrate to remove partial alcohol and water to obtain crude extract;
the resin purification step comprises loading the crude extract into an adsorption resin column, and sequentially performing water elution and gradient analysis of an alcohol-water solution to obtain an eluent;
a decoloring step, which comprises the steps of adsorbing and decoloring eluent by using active carbon and filtering to obtain decoloring liquid;
concentrating and drying, namely concentrating and drying the decolorized solution to obtain a powdery bee grass extract.
According to an embodiment of one aspect of the present application, in the mixed solution of alcohol and water, the mass fraction of alcohol in the mixed solution is 50-80%.
According to an embodiment of one aspect of the present application, the alcohol comprises one or more of ethanol, ethylene glycol, glycerol, 1, 2-butanediol.
According to an embodiment of one aspect of the application, at least one of the following process conditions is fulfilled in the extracting step:
1) The ratio of the crushed materials of the melissa officinalis to the mixed solution is 1: (10-30);
2) The extraction temperature is 40-90 ℃;
3) The extraction time is 2-10 h;
4) The number of extractions is at least one;
5) At least one of stirring extraction, solvent extraction and ultrasonic extraction is adopted for extraction.
According to an embodiment of one aspect of the present application, the filtering step includes filtering the crude extract with a filter membrane under a condition of negative pressure suction filtration or positive pressure filtration to obtain a filtrate.
According to an embodiment of one aspect of the application, the pore size of the filter membrane is 0.22 μm to 10 μm.
According to an embodiment of one aspect of the application, the mass content of the ethanol in the crude extract is less than or equal to 10%.
According to an embodiment of one aspect of the present application, the adsorption resin comprises at least one of D101 type macroporous adsorption resin, AB-8 type macroporous adsorption resin and HPD100 type macroporous adsorption resin.
According to an embodiment of one aspect of the application, the water-eluting water usage is 3 BV-5 BV.
According to an embodiment of one aspect of the application, the gradient analysis of the aqueous alcohol solution comprises: eluting with alcohol water solution of 20-30% mass fraction at 1.5-2BV/h, eluting with alcohol water solution of 4-5BV, receiving the first eluent, eluting with alcohol water solution of 40-50% mass fraction at 2-3 BV, discarding the eluent, eluting with alcohol water solution of 80-90% mass fraction at 4-5BV, receiving the second eluent, and mixing the first eluent and the second eluent to obtain the eluent.
According to an embodiment of one aspect of the application, the decolorizing step satisfies at least one of the following process conditions:
the addition amount of the activated carbon accounts for 1-5% of the mass of the eluent;
the decoloring time is 20-60 min;
the decoloring temperature is 30-70 ℃.
According to an embodiment of one aspect of the application, the concentrating and drying steps meet any one of the following process conditions:
1) Drying by using a spray dryer;
optionally, the inlet temperature of the spray dryer is 100-160 ℃, and the outlet temperature is 60-90 ℃;
optionally, the feeding speed of the concentrated decolorized solution in a spray dryer is 5-40 mL/min;
optionally, drying the concentrated decolorized solution by using a spray dryer under the condition that the compressed air pressure is 0.4-0.8 MPa;
2) The drying is carried out using a granulator, optionally at a drying temperature of 105 to 125 ℃.
In a second aspect, embodiments of the present application provide a melissa extract prepared by the method of the first aspect.
According to an embodiment of one aspect of the present application, the melissa extract comprises flavonoids, rosmarinic acid, caffeic acid.
According to an embodiment of one aspect of the application, the mass content of total flavonoids in the melissa officinalis extract is 40% -50%, the mass content of luteolin is 5% -10%, the mass content of rosmarinic acid is 20% -25%, and the mass content of caffeic acid is 2% -3%.
According to an embodiment of one aspect of the application, the coumarin content of the melissa extract is below the detection limit, further optionally < 4 μg/ml.
According to an embodiment of an aspect of the present application, the melissa extract has at least one of sun-screening, anti-glycation, collagen increase promotion, dermis epidermis junction promotion, and soothing effects.
In a third aspect, embodiments of the present application provide a cosmetic composition comprising a melissa extract prepared by the method of the first aspect or the melissa extract of the second aspect.
According to an embodiment of an aspect of the present application, the melissa extract is 0.05% -10% by mass in the cosmetic composition.
Compared with the prior art, the application has at least the following beneficial effects:
the method provided by the application can prepare the nano-powder-like melissa officinalis extract, and the coumarin content in the melissa officinalis extract is lower than the detection limit. In the method, the total flavone component is selectively collected through a resin purification process, so that the mass content of the total flavone component is improved to 40% -50%; the active ingredients are selectively collected by a resin purification process, the active ingredients of which are determined, and luteolin: rosmarinic acid: the caffeic acid selectively removes coumarin substances in the melissa officinalis extract by matching the resin purification process and the related process, and ensures the efficacy and safety of the melissa officinalis extract.
The cosmetic composition containing the melissa extract has good effects of sun protection, anti-saccharification, promotion of collagen increase, promotion of dermal epidermis connection and relaxation, and has good application prospect in the aspect of cosmetics.
Drawings
In order to more clearly illustrate the technical solution of the embodiments of the present application, the drawings that are needed in the embodiments of the present application will be briefly described, and other drawings can be obtained according to these drawings without inventive effort for a person skilled in the art.
Fig. 1 is a schematic flow chart of a method for preparing a melissa extract provided in example 1 of the present application.
Detailed Description
In order to make the application object, technical scheme and beneficial technical effects of the application clearer, the application is further described in detail with reference to the following embodiments. It should be understood that the embodiments described in this specification are for the purpose of illustrating the application only and are not intended to limit the application.
For simplicity, only a few numerical ranges are explicitly disclosed. However, any lower limit may be combined with any upper limit to form a range not explicitly recited; and any lower limit may be combined with any other lower limit to form a range not explicitly recited, and any upper limit may be combined with any other upper limit to form a range not explicitly recited. Furthermore, each point or individual value between the endpoints of the range is included within the range, although not explicitly recited. Thus, each point or individual value may be combined as a lower or upper limit on itself with any other point or individual value or with other lower or upper limit to form a range that is not explicitly recited.
In the description of the present application, unless otherwise indicated, "above" and "below" are intended to include the present number, and the meaning of "multiple" in "one or more" means two or more.
The above summary of the present application is not intended to describe each disclosed embodiment or every implementation of the present application. The following description more particularly exemplifies illustrative embodiments. Guidance is provided throughout this application by a series of embodiments, which may be used in various combinations. In the various examples, the list is merely a representative group and should not be construed as exhaustive.
Method for preparing herba Melissae axillaris extract
In a first aspect, embodiments of the present application provide a method for preparing a melissa extract, comprising:
crushing, namely crushing the raw materials of the melissa officinalis to obtain crushed melissa officinalis;
the extraction step comprises extracting the crushed herba Melissae axillaris with a mixed solution of alcohol and water to obtain a crude extract;
filtering, namely filtering the crude extract by adopting a filter membrane to obtain filtrate;
concentrating, namely vacuum concentrating the filtrate to remove partial alcohol and water to obtain crude extract;
The resin purification step comprises loading the crude extract into an adsorption resin column, and sequentially performing water elution and gradient analysis of an alcohol-water solution to obtain an eluent;
a decoloring step, which comprises the steps of adsorbing and decoloring eluent by using active carbon and filtering to obtain decoloring liquid;
concentrating and drying, namely concentrating and drying the decolorized solution to obtain a powdery bee grass extract.
According to the embodiment of the application, the raw materials of the melissa officinalis can be melissa officinalis dry materials or wet materials with certain moisture. The raw materials of melissa are to be understood broadly and include raw materials of melissa. The raw materials of the melissa officinalis are crushed into powder, and the powder can be screened by a screen to obtain uniform melissa officinalis crushed materials. The decolorized solution can be clear and yellow brown solution containing herba Melissae axillaris extract; the powdered bee grass extract can be nanometer grade, and has particle diameter of about 50nm-200nm.
Through researches, the melissa extract can exert the effects of repairing skin barrier by reducing percutaneous moisture loss, reducing erythema index, increasing skin hydration value and the like; the herba Melissae axillaris extract can improve the activity of keratinocytes under irradiation of hydrogen peroxide and UVB; citral in herba Melissae axillaris extract can inhibit TNF-alpha in RAW 264.7 cell stimulated by Lipopolysaccharide (LPS), and inhibit IL-6 and IL-1β in peritoneal macrophages stimulated by normal mouse LPS, and has antiinflammatory effect.
Further researches show that the melissa extract has good effects of sun protection, anti-saccharification, promotion of collagen increase, promotion of dermal epidermis connection and relief, and has good application prospect in the aspect of cosmetics.
According to the embodiment of the application, the method has the advantages of simple process, low cost, no toxic or harmful solvent, recoverability, short time consumption, low equipment requirement, good compatibility with the existing process and great large-scale application potential.
In some embodiments, the mass fraction of the alcohol in the mixed solution of the alcohol and the water is 50-80%. The alcohol concentration and the ratio of water in the mixed solution may affect the solubility and extraction efficiency of the extract. Alcohol-water mixtures of different concentrations may have different solubilities for the active ingredients in melissa officinalis, thereby affecting the extraction effect. The active ingredients in the melissa officinalis can be fully extracted by controlling the mass fraction in the mixed solution in the range, so that the yield of the extract is improved.
In some embodiments, the alcohol comprises one or more of ethanol, ethylene glycol, glycerol, 1, 2-butanediol. The alcohol is nontoxic and safe, and is easy to mix with water. After the alcohol is mixed with water, the active ingredients in the melissa officinalis can be fully extracted, and the yield of the extract is improved.
In some embodiments, at least one of the following process conditions is met in the extracting step:
1) The ratio of the crushed materials of the melissa officinalis to the mixed solution is 1: (10-30);
2) The extraction temperature is 40-90 ℃;
3) The extraction time is 2-10 h;
4) The number of extractions is at least one;
5) At least one of stirring extraction, solvent extraction and ultrasonic extraction is adopted for extraction.
According to the embodiment, the ratio of the crushed materials of the melissa officinalis to the mixed solution can affect the solubility and extraction efficiency of the extract. Different feed-to-liquid ratios may have different solubilities for the active ingredients in the melissa, thereby affecting the extraction effect. The active ingredients in the melissa officinalis can be fully extracted by controlling the feed liquid ratio in the range, so that the yield of the extract is improved.
According to embodiments per se, the extraction temperature during the extraction process may affect the chemical reaction and the component release rate in the extract. Different temperature conditions may lead to different extraction effects and chemical changes, in which temperature ranges the desired components can be further obtained and the relevant unwanted components removed.
According to embodiments per se, the length of time in the extraction process may affect the concentration and composition of the extract. Longer extraction times may result in more active ingredient being released from the melissa, but too long a time may result in extraction of other unwanted ingredients. The melissa extract with proper composition is prepared by controlling the extraction time in the above range.
In some embodiments, the filtering step comprises filtering the crude extract with a filter membrane under conditions of negative pressure suction filtration or positive pressure filter filtration to obtain a filtrate.
According to the embodiment of the application, the crude extract is filtered by using the filter membrane under the conditions of negative pressure suction filtration or positive pressure filter pressing, so that the quality and purity of the extract can be influenced. The following are possible influencing factors:
in some embodiments, the pore size of the filter is 0.22 μm to 10 μm. According to embodiments of the present application, the pore size and selectivity of the membrane may affect filtration efficiency. The pore diameter can effectively prevent solid particles or impurities from passing through the filter membrane, and the filter efficiency is improved.
In some embodiments, the mass content of ethanol in the crude extract is less than or equal to 10%. According to the embodiment of the application, the mass content of the ethanol in the crude extract is controlled within the range, so that the concentration degree is controlled, and the subsequent adsorption and decolorization are facilitated.
In some embodiments, the adsorbent resin comprises at least one of D101 type macroporous adsorbent resin, AB-8 type macroporous adsorbent resin, HPD100 type macroporous adsorbent resin.
The D101 macroporous adsorption resin is polystyrene based adsorption resin. It has large pore size and surface area, and is used in extracting Chinese medicine, biological medicine and food industry. The D101 type adsorption resin can separate target compounds such as active ingredients in the extract, enzymes, etc. by selective adsorption.
AB-8 type macroporous adsorption resin: AB-8 type macroporous adsorbent resin is a polystyrene-based strong polar adsorbent resin. The AB-8 type adsorption resin has larger aperture and stronger hydrophilicity, and can remove heavy metal ions, pigments and the like in water.
The HPD100 type macroporous adsorption resin is a polystyrene based nonionic adsorption resin. It has larger pore diameter and higher adsorption capacity, and is commonly used for protein purification, enzyme separation and purification and adsorption separation of other macromolecular compounds.
According to the embodiment of the application, the adsorption resin of the type can selectively adsorb and separate different molecular characteristics according to the specific physicochemical properties and structural design, and can adsorb part of harmful substances in the active ingredients of the melissa officinalis on the basis of retaining the active ingredients of the melissa officinalis to the greatest extent, thereby realizing separation and purification. Certain components may also be selectively separated or retained, such as coumarin or the like, to alter the composition of the extract.
In some embodiments, the water is used in an amount of 3BV to 5BV.
According to an embodiment of the present application, water elution refers to a process of washing and removing impurities or non-target compounds adsorbed on a resin using an aqueous solution. By using the above amount of water, non-specific impurities, ions or low affinity compounds adsorbed on the resin can be effectively removed, and impurities, residues and redundant reagents on the surface of the resin can be removed to purify the resin and restore the adsorption performance thereof, thereby improving the purity of the target compound.
In some embodiments, the gradient resolution of the aqueous alcohol solution comprises: eluting with alcohol water solution of 20-30% mass fraction at 1.5-2BV/h, eluting with alcohol water solution of 4-5BV, receiving the first eluent, eluting with alcohol water solution of 40-50% mass fraction at 2-3 BV, discarding the eluent, eluting with alcohol water solution of 80-90% mass fraction at 4-5BV, receiving the second eluent, and mixing the first eluent and the second eluent to obtain the eluent.
According to an embodiment of the present application, gradient analysis of an aqueous alcohol solution refers to the use of solutions of different concentrations of alcohol and water to gradually elute a target compound adsorbed on a resin. The affinity of the resin to the target compound can be changed by controlling the gradient analysis of the alcohol aqueous solution and adjusting the proportion and the concentration of the alcohol and the water in the range, so that the selective adsorption and desorption of different compounds are realized, the target compound is further obtained, and the harmful compounds are removed, such as flavonoid compounds, rosmarinic acid and caffeic acid are obtained, and coumarin is removed; the polarity and elution capacity of the solution can be gradually changed by controlling the gradient analysis of the alcohol aqueous solution, so that the compounds adsorbed on the resin are gradually eluted, and the purification of the target compounds is realized.
In some embodiments, the decolorizing step satisfies at least one of the following process conditions:
the addition amount of the activated carbon accounts for 1-5% of the mass of the eluent;
the decoloring time is 20-60 min;
the decoloring temperature is 30-70 ℃.
According to the embodiment of the application, the activated carbon has good adsorption performance, and can effectively remove pigments, impurities, peculiar smell substances and the like in the solution. The method has the advantages that the active carbon is used for decoloring, the related process conditions of decoloring are controlled, the clarity and purity of the solution can be improved, and the solution can be effectively decolored and purified, so that the solution meets specific quality standards and application requirements.
In some embodiments, the concentrating and drying steps satisfy any one of the following process conditions:
1) Drying by using a spray dryer;
optionally, the inlet temperature of the spray dryer is 100-160 ℃, and the outlet temperature is 60-90 ℃;
optionally, the feeding speed of the concentrated decolorized solution in a spray dryer is 5-40 mL/min;
optionally, drying the concentrated decolorized solution by using a spray dryer under the condition that the compressed air pressure is 0.4-0.8 MPa;
2) The drying is carried out using a granulator, optionally at a drying temperature of 105 to 125 ℃.
According to embodiments of the present application, controlling the temperature, time, and feed rate during concentration and drying may affect the morphology, particle size distribution, and physical properties of the nanoscale powder. Controlling the inlet and outlet temperatures of the spray dryer can affect the morphology, particle size distribution of the nanoscale powders. In the temperature range, on the premise of considering the drying speed, enough heat energy can be provided to accelerate the evaporation of water and the drying speed of solid particles, so that the agglomeration, sintering or thermal decomposition of powder possibly caused by the excessive temperature is avoided. The drying time can be controlled by controlling the feeding speed of the concentrated decolorized solution, so that the complete evaporation of the solvent can be ensured, the powder can reach the required drying degree, and excessive drying and powder agglomeration are avoided. In the drying process, the pressure of the compressed air is controlled to be 0.4-0.8 MPa, so that the proper environment in the drying equipment can be kept, uniform drying of the powder is facilitated, and uneven drying and agglomeration of the powder can be avoided.
According to the embodiment of the application, the process conditions are optimized, so that higher extraction efficiency, better extract quality and higher yield can be realized.
In a second aspect, embodiments of the present application provide a melissa extract prepared by the method of the first aspect.
In some embodiments, the melissa extract comprises flavonoids, rosmarinic acid, caffeic acid.
In some embodiments, the mass content of total flavonoids in the melissa extract is 40% -50%, the mass content of luteolin is 5% -10%, the mass content of rosmarinic acid is 20% -25%, and the mass content of caffeic acid is 2% -3%.
In some embodiments, the mass ratio of luteolin, rosmarinic acid, caffeic acid is 1: (4-5): (0.4-0.6).
In some embodiments, the coumarin content of the Melissa officinalis extract is below the detection limit, further optionally < 4 μg/ml.
In some embodiments, the melissa extract has at least one of sun protection, anti-glycation, collagen increase promotion, dermis epidermis junction promotion, soothing.
In a third aspect, embodiments of the present application provide a cosmetic composition comprising a melissa extract prepared by the method of the first aspect or the melissa extract of the second aspect.
According to the embodiment of the application, the cosmetic composition is safe and non-irritating, and has good sun-screening effect, anti-saccharification effect, collagen increasing effect and dermis epidermis connection effect. The cosmetic composition can be used for preparing cosmetic water, facial cleanser, lotion, facial cream, essence, etc.
In some embodiments, the melissa extract is 0.05% -10% by mass of the cosmetic composition.
According to the embodiment of the application, the quality fraction of the melissa officinalis extract in the cosmetic composition is controlled within the above range, so that the cosmetic composition is beneficial to further ensuring the good sun-proof effect, anti-saccharification effect, collagen increase promoting effect and dermis epidermis connection promoting effect.
In some embodiments, the lemon balm cream comprises the following components in mass percent:
| component (A) | The mass percentage content |
| Deionized water | Allowance of |
| Glycerol | 4-6 |
| Sodium hyaluronate | 0.05-0.2 |
| Xanthan gum | 0.15-0.3 |
| 1, 3-butanediol | 2-4 |
| Caprylic/capric triglyceride | 4-6 |
| Cetostearyl alcohol | 2-4 |
| Jojoba seed oil | 2-4 |
| Bush fruit resin | 1-2.8 |
| Coco-caprylate/caprate | 1-2.8 |
| Glycerol stearate and PEG-100 stearate | 2-4 |
| Melissa officinalis extract | 0.15-0.3 |
| Tocopheryl acetate | 0.08-0.15 |
| Preservative agent | 0.8-2 |
| Citric acid | 0.04-0.06 |
。
In some embodiments, the lemon balm cream comprises the following components in mass percent:
| component (A) | The mass percentage content |
| Deionized water | Allowance of |
| Glycerol | 5 |
| Sodium hyaluronate | 0.1 |
| Xanthan gum | 0.2 |
| 1, 3-butanediol | 3 |
| Caprylic/capric triglyceride | 5 |
| Cetostearyl alcohol | 3 |
| Jojoba seed oil | 3 |
| Bush fruit resin | 2 |
| Coco-caprylate/caprate | 2 |
| Glycerol stearate and PEG-100 stearate | 3 |
| Melissa officinalis extract | 0.2 |
| Tocopheryl acetate | 0.1 |
| Preservative agent | 1 |
| Citric acid | 0.05 |
。
Examples
The present disclosure is more particularly described in the following examples that are intended as illustrations only, since various modifications and changes within the scope of the present disclosure will be apparent to those skilled in the art. Unless otherwise indicated, all parts, percentages, and ratios reported in the examples below are by weight, and all reagents used in the examples are commercially available or were obtained synthetically according to conventional methods and can be used directly without further treatment, and the instruments used in the examples are commercially available.
Example 1
The embodiment provides a preparation method of a melissa extract, which comprises the following steps:
pulverizing herba Melissae axillaris in pulverizer, sieving with 40 mesh sieve, collecting 100g powder, adding 2000mL 70% ethanol solution, stirring at 80deg.C at 200r/min for 2 hr, and extracting for 2 times to obtain herba Melissae axillaris crude extract; vacuum concentrating the obtained filtrate with a filter membrane aperture of 5 μm, controlling the concentration temperature to be lower than 50 ℃ until no alcohol smell exists, obtaining extract, loading the obtained extract into a D101 macroporous resin column, eluting with water (the water washing flow rate is 2BV/h, the eluting volume is 5 BV), then resolving with 20% ethanol solution, eluting the eluting volume is 5BV, collecting the resolved solution, eluting with 40% ethanol water solution for 3BV, discarding the part of the eluent, eluting with 80% ethanol water solution for 5BV, collecting the eluent, mixing the collected eluent, adding 1% active carbon, stirring and decolorizing at 50 ℃ for 30min, vacuum filtering, concentrating the filter membrane aperture of 1 μm to 100mL, granulating with a spray dryer, the inlet temperature of the spray dryer is 160 ℃, the outlet temperature is 90 ℃, the feeding speed is 5mL/min, and the compressed air pressure is 0.4MPa, thus obtaining nano-level melissa extract powder, about 9.5 g.
Example 2
In this example, compared with example 1, the resin filler type was changed to AB-8, and the other conditions were unchanged.
Example 3
This embodiment differs from embodiment 1 in that: the extraction temperature was 65 ℃.
Example 4
This embodiment differs from embodiment 1 in that: the pore size of the filter membrane was 4. Mu.m.
Example 5
This embodiment differs from embodiment 1 in that: gradient resolution of aqueous alcohol solutions differs, comprising: eluting with alcohol water solution at an eluting speed of 1.8BV/h, eluting with 25% alcohol water solution at a mass fraction of 4.2BV, receiving the first eluent, eluting with 45% alcohol water solution at a mass fraction of 2.5BV, discarding the eluent, eluting with 85% alcohol water solution at a mass fraction of 4.5BV, receiving the second eluent, mixing the first eluent and the second eluent, and obtaining the eluent.
Example 6
This embodiment differs from embodiment 1 in that: gradient resolution of aqueous alcohol solutions differs, comprising: eluting with 28% ethanol water solution at an eluting speed of 2.0BV/h, eluting with 4.2BV, receiving the first eluent, eluting with 44% ethanol water solution at a mass fraction of 2.4BV, discarding the eluent, eluting with 85% ethanol water solution at a mass fraction of 4.2BV, receiving the second eluent, mixing the first eluent and the second eluent, and collecting the eluent.
Comparative example 1
In this comparative example, compared with example 1, the resin elution process was changed to water elution of 5BV, gradient analysis of the aqueous alcohol solution was different, elution was performed with 50% aqueous alcohol solution of 5BV, and the 50% aqueous alcohol solution eluent was collected under the same conditions.
Comparative example 2
In this comparative example, the extraction solvent was changed to a 10% ethanol aqueous solution, and the other conditions were unchanged from example 1.
Test part
1) Detection of total flavone content in herba Melissae axillaris extract
The Melissa officinalis extracts obtained in example 1, example 2, comparative example 1 and comparative example 2 were subjected to determination of total flavone content
And (3) taking rutin as a standard sample, and measuring the total flavone content in the obtained melissa powder by adopting a color development method.
(1) Drawing a standard curve, precisely measuring 0.108mg/mL of rutin reference substance, preparing a series of concentrations according to Table 1, developing for half an hour, and detecting absorbance at a wavelength of 510 nm.
TABLE 1
(2) Assay of samples to be tested the melissa extract powder of example 1, example 2, comparative example 1, comparative example 2 was formulated as 0.5% melissa extract, and 0.5mL was diluted to 10mL. Taking 0.5mL of liquid to be detected, respectively adding 0.3mL of 5% sodium nitrite, 0.3mL of 10% aluminum nitrate, 6.9mL of 30% ethanol and 3mL of 1mol/L sodium hydroxide, developing for half an hour, and carrying out concentration measurement to obtain the total flavone content shown in Table 2:
TABLE 2
2) Detection of luteolin content in Melissa officinalis extract
Determination of luteolin content by Melissa powder obtained in example 1, example 2, comparative example 1, comparative example 2
(1) HPLC chromatographic conditions: the mobile phase is acetonitrile-0.1% phosphoric acid aqueous solution (20:80); chromatographic column: c18 5 μm (4.6 mm x 250 mm); detection wavelength: 330nm; column temperature:
25 ℃; flow rate: 1mL/min.
(2) Control test material: standards were purchased from Shanghai microphone Biochemical technologies Co. 6mg of rosmarinic acid (HPLC is more than or equal to 98%) is taken, 50% ethanol is added to prepare 120 mug/mL of reference substance solution, and 0, 5, 10, 15, 20, 30 and 40 mug of reference substance solution are respectively injected to obtain a linear curve.
(3) Test solution: 0.5g of the melissa powder of example 1, example 2, comparative example 1 and comparative example 2 is dissolved in 100mL of 50% ethanol solution to obtain 0.5% melissa extractive solution, the sample injection amount is 10 mu L, and the luteolin content in the melissa powder is measured as shown in Table 3:
TABLE 3 Table 3
3) Rosmarinic acid content detection of melissa officinalis extract
Quantitative analysis of rosmarinic acid component on Melissa officinalis extract obtained in example 1, example 2, comparative example 1, comparative example 2
(1) HPLC chromatographic conditions: the mobile phase is methanol-0.1% phosphoric acid aqueous solution (50:50); chromatographic column: c18 5 μm (4.6 mm x 250 mm); detection wavelength: 350nm; column temperature: 25 ℃;
Flow rate: 1mL/min.
(2) Control test material: standards were purchased from Shanghai microphone Biochemical technologies Co. 6mg of rosmarinic acid (HPLC is more than or equal to 97%) is taken, 50% ethanol is added to prepare 120 mug/mL of reference substance solution, and 0, 5, 10, 15, 20, 30 and 40 mug of reference substance solution are respectively injected to obtain a linear curve.
(3) Test solution: in example 1, example 2, comparative example 1 and comparative example 2, 0.5g of the melissa powder was dissolved in 100mL of 50% ethanol solution to obtain 0.5% melissa extract, 1mL of 0.5% melissa extract was taken, 50% ethanol was added to dilute to 10mL, the sample injection amount was 10 μl, and the rosmarinic acid content in the melissa powder was measured as shown in table 4:
TABLE 4 Table 4
4) Detection of caffeic acid content in melissa officinalis extract
The melissa extract obtained in example 1, example 2, comparative example 1 and comparative example 2 was subjected to caffeic acid component quantitative analysis
(1) HPLC chromatographic conditions: the mobile phase was methanol-phosphate buffer (25:75, ph=2.3);
chromatographic column: c18 5 μm (4.6 mm x 150 mm); detection wavelength: 323nm; column temperature:
25 ℃; flow rate: 0.8mL/min.
(2) Control test material: standards were purchased from Shanghai microphone Biochemical technologies Co. 5mg of caffeic acid (HPLC is more than or equal to 97%) is taken, 50% ethanol is added to prepare a reference substance solution with the concentration of 50 mug/mL, and 0, 5, 10, 15, 20, 30 and 40 mug are respectively injected to obtain a linear curve.
(3) Test solution: examples 1, 2, comparative example 1 and comparative example 2 were prepared by dissolving 0.5g of the melissa powder in 100mL of 50% ethanol solution to obtain 0.5% melissa extract, adding 1mL of 0.5% melissa extract to 10mL of 50% ethanol, diluting to a sample size of 10. Mu.L, and measuring the caffeic acid content in the melissa powder as shown in Table 5:
TABLE 5
5) Coumarin content detection of melissa officinalis extract
Coumarin component analysis was performed on the melissa extract obtained in example 1, example 2, comparative example 1 and comparative example 2 the content of coumarin in the melissa extract powder in example 1, example 2, comparative example 1 and comparative example 2 was measured by referring to "high performance liquid chromatography for determination of coumarin and derivatives thereof in cosmetics" GB/T35798-2018 ", and the content of coumarin in the melissa extract powder was measured as shown in Table 6:
the following is noted: the detection limit of the method is 4 mug/ml
TABLE 6
6) Cytotoxicity test
Immortalized keratinocytes were cultured in a culture medium containing 10% fetal bovine serum and 1% diabody (1X 10) 5 U/L penicillin, 100mg/L streptomycin) in DMEM medium. Cell growth at 37℃with 5% CO 2 Culturing in an incubator until the cell fusion degree is 85-95%; digesting cells in logarithmic growth phase with pancreatin at a concentration of 0.05% by weight, and administering a pharmaceutical composition comprising DMEM medium with 10% serum stopped the digestion reaction. Counting with a cell counting plate, adjusting the cell suspension to 2 x 10≡5/ml, inoculating to 96-well plate at a ratio of 200 μl per well, and inoculating at 37deg.C with 5% CO 2 Incubating for a certain time under the condition until the cell fusion degree is 45-60%; the old medium was removed, 200. Mu.L of serum-free medium containing samples to be tested of Melissa extract at different concentrations was added, and 4 wells were made per test solution. Blank cell-free, 200 μl PBS was added; example combinations the comparative example group contains cells and 200 μl of serum free medium is added. At 37℃, 5% CO 2 Incubating for 24 hours under the condition; then adding 20 mu L of CCK8 solution into each hole, incubating for 3 hours, measuring absorbance value at 450nm, and calculating the cell viability of each group;
1. cell viability (%) = (sample group-blank)/(control group-blank) ×100;
2. the final concentrations of the Melissa extract in examples 1-2 and comparative examples 1-2 were 12.5. Mu.g/ml, 25. Mu.g/ml, 50. Mu.g/ml, 100. Mu.g/ml, 250. Mu.g/ml in the following samples.
The results of the horniness cell toxicity test of the melissa extract are shown in table 7:
TABLE 7
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Note that: in this experiment, the higher the cell viability, the less cytotoxic the extract of melissa is.
The experimental results show that: is a negative control group; the Melissa officinalis extracts of examples 1 and 2 were both non-cytotoxic, and at high concentrations, keratinocytes were normal in morphology and the relative cell viability increased to around 130%, with comparative example 1 being slightly cytotoxic at high concentrations, and comparative example 2 being non-cytotoxic.
8) Cell proliferation assay
1. Immortalized horny substanceThe osteoblasts were cultured in DMEM medium containing 10% fetal bovine serum and 1% diabody (penicillin 1X 105U/L, streptomycin 100 mg/L). Cell growth at 37℃with 5% CO 2 Culturing in an incubator until the cell fusion degree is 85-95%;
2. cells in the logarithmic growth phase were digested with pancreatin at a concentration of 0.05% and the digestion was stopped with DMEM medium containing 10% serum.
3. Counting with a cell counting plate, adjusting the cell suspension to 2 x 10≡5/ml, inoculating to 96-well plate at a ratio of 200 μl per well, and inoculating at 37deg.C with 5% CO 2 Incubating for a certain time under the condition until the cell fusion degree is 45-60%;
4. the old medium was removed, 100. Mu.L of serum-free medium containing samples to be tested of Melissa officinalis extract was added, and 4 wells were made per liquid to be tested. Blank cell-free, 200 μl PBS was added; EXAMPLE combination comparative example groups contained cells in 100. Mu.L of serum-free medium at 37℃in 5% CO 2 Incubating for 24 hours under the condition;
5. then adding 20 mu L of CCK8 solution into each hole, incubating for 3 hours, measuring absorbance value at 450nm, and calculating the cell viability of each group;
6. cell viability (%) = (sample group-blank)/(control group-blank) ×100;
7. raw material test examples 1-2, the final concentrations of the Melissa officinalis extract in comparative examples 1-2 were 250 μg/ml, respectively;
the test results are shown in table 8:
TABLE 8
| 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 |
Conclusion of experiment: examples 1 and 2 have the effect of promoting keratinocyte proliferation at a test concentration of 250. Mu.g/ml.
9) UV damaging keratinocyte assay
1. Immortalized keratinocytes were cultured in DMEM medium containing 10% fetal bovine serum and 1% diabody (penicillin 1X 105U/L, streptomycin 100 mg/L). Cell growth at 37℃with 5% CO 2 Culturing in an incubator until the cell fusion degree is 85-95%;
2. cells in the logarithmic growth phase were digested with pancreatin at a concentration of 0.05% and the digestion was stopped with DMEM medium containing 10% serum.
3. Cell suspensions were adjusted to 2 x 10≡5/ml by counting with a cell counting plate and seeded in 96-well plates at a rate of 100 μl per well. At 37℃with 5% CO 2 Incubating for a certain time under the condition until the cell fusion degree is 85-95%;
4. the old medium was removed, 100. Mu.L of serum-free medium containing samples to be tested of Melissa officinalis extract was added, and 4 wells were made per liquid to be tested. Blank cell-free, 200 μl PBS was added; the control group contained cells, 100. Mu.L of serum-free medium was added thereto, and the mixture was subjected to 5% CO at 37 ℃ 2 Incubating for 24h under the condition, wherein the final concentration of the herba Melissae axillaris extract in the sample to be tested in examples 1-2 and comparative examples 1-2 is 250 mug/m respectivelyl；
5. By 8J/cm 2 Control cells and dosing cells.
6. Then adding 20 mu L of CCK8 solution into each hole, incubating for 3 hours, measuring absorbance value at 450nm, and calculating the cell viability of each group;
the test results are shown in table 9:
TABLE 9
| Project | 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 |
Conclusion of experiment: the Melissa officinalis extract of example 1 and example 2 has protective effect on UVA damaged keratinocytes at a test concentration of 250 μg/ml.
3) ELISA histamine assay
The ELISA histamine kit was derived from Biotechnology Co.Ltd.
1. Sample addition of standard substance: standard wells and sample wells were set, and 50 μl of standard of different concentrations was added to each standard well according to the kit instructions.
2. Sample adding: blank holes (blank control holes are not added with samples and enzyme-labeled reagents, and the rest steps are the same) and sample holes to be tested are respectively arranged. The sample dilution liquid is added into 40 mu L of the sample to be detected in the sample hole on the enzyme-labeled coated plate, and then 10 mu L of the sample to be detected is added (the final dilution of the sample is 5 times). And (3) adding a sample to the bottom of the ELISA plate hole, so as not to touch the hole wall as much as possible, and slightly shaking and uniformly mixing. Examples 1-2, comparative examples 1-2 each had a final concentration of the Melissa officinalis extract of 250. Mu.g/ml.
3. Adding enzyme: 100. Mu.L of enzyme-labeled reagent was added to each well, except for blank wells.
4. Incubation: the plates were then covered with a plate membrane and incubated at 37℃for 60 minutes.
5. Preparing liquid: the 20-fold concentrated washing solution is diluted with distilled water for later use.
6. Washing: carefully removing the sealing plate film, discarding the liquid, spin-drying, filling each hole with the washing liquid, standing for 30 seconds, discarding, repeating the process for 5 times, and beating.
7. Color development: 50 mu L of a color developing agent A and 50 mu L of a color developing agent B are added into each hole, the mixture is gently vibrated and mixed uniformly, and the color is developed for 15 minutes at 37 ℃ in a dark place.
8. And (3) terminating: the reaction was stopped by adding 50. Mu.L of stop solution to each well (blue turned yellow immediately).
9. And (3) measuring: absorbance (OD) was measured sequentially for each well using a blank Kong Diaoling, 450nm wavelength. The measurement should be performed within 15 minutes after the addition of the stop solution.
Table 10
| 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 |
Conclusion of experiment: the Melissa officinalis extract of example 1 and example 2 can significantly inhibit release of mast cell histamine at a test concentration of 250 μg/ml, so that allergic symptoms can be alleviated and can be alleviated, irritation and damage of histamine to skin can be reduced, and the skin health can be improved.
11 Inhibition test of inflammatory factor
1. Cell inoculation: RAW264.7 cells (North Naiocyte library; epiSkin, shanghai An Funuo Biotech Co., ltd.) were seeded at 1.5X105 into 24-well plates at 37℃with 5% CO 2 Incubating the incubator overnight;
2. experimental grouping: setting a blank control group, a negative control group and a sample group respectively, wherein the blank control group, the negative control group and the sample group comprise examples 1-2 and comparative examples 1-2;
3. preparing liquid: preparing a sample working solution according to a test concentration setting table;
4. sample feeding: and (5) feeding samples when the cell plating rate of the 24-well plate reaches 50-60%. Adding a medium containing LPS into each negative control group; sample groups (examples 1-2 and comparative examples 1-2) each had a medium containing LPS and samples of corresponding concentrations added thereto, and the final concentrations of the Melissa officinalis extract in the final samples of examples 1-2 and comparative examples 1-2 were 50. Mu.g/ml, respectively; adding a blank or solvent control group into the culture medium without LPS;
5. After the completion of the administration, the 24-well plate was placed in an incubator (37 ℃, 5% CO) 2 ) Culturing in medium; observing the cell morphology under a microscope and taking a picture;
6. and (3) detection: after incubation of the cells for 24h, 500. Mu.l of the cell supernatant was collected for determination of the inflammatory factor NO content. Adding a DMEM diluted CCK8 solution, incubating at 37 ℃, and reading an OD value at 450 nm;
7. cell relative viability (%) = (sample group OD-zeroed group OD)/(solvent control group OD-zeroed group OD) ×100;
8. the test results are shown in Table 11
TABLE 11
| Project | Release amount (mu M) |
| 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 example2 | 23.15±0.21* |
The experimental results show that: after RAW264.7 cells are stimulated by LPS, the excessive expression NO reaches 28.59 mu M; after the treatment of the melissa extract of the embodiment 1 and the embodiment 2 with the concentration of 50ug/mL, the expression level of the mouse macrophage NO is obviously reduced, which indicates that the melissa extract can well act on RAW264.7 cells stimulated by LPS, so that the inflammatory reaction of the cells can be well relieved.
12 Anti-glycation effect detection
The experimental method comprises the following steps: 18.22g Balb/c mice (SPF grade), males, purchased from Experimental animal technologies Inc. of Beijing Vitre. A diabetic mouse model was established to study the effects of different analytes on AGE in organs such as heart, liver and kidney. Mice were intravenously injected with tetraoxapyrimidine (80 mg/kg) at the tail of the mice to establish a diabetic mouse model. 50 eligible mice were selected and randomly divided into model and experimental groups (example 1, comparative example 2), 10 each. 10 healthy mice were also taken as a blank group, which did not construct a diabetic mouse model, nor did gastric lavage. The model group and the experimental group were respectively perfused with physiological saline and the extracts of Melissa officinalis of example 1, example 2, comparative example 1 and comparative example 2, and the respective intragastric concentrations were 30mg/kg (physiological saline was used as a solvent in the experimental group), once daily for 14 days. Mice were fasted for 16 hours after the last dose, and then cervical spine removed for sacrifice for subsequent experiments. Mouse heart, liver and kidney tissues were cut into small pieces, homogenized and centrifuged. To the precipitate was added 5mL chloroform methanol (2:1) for degreasing and shaking overnight at 4 ℃. Then, after washing in HEPES buffer and overnight, collagenase was added and digested at 37℃for 24 hours. The above solution was centrifuged to obtain a supernatant, and the fluorescence intensity was measured at 370nm/440nm using a fluorescence spectrophotometer. The relative fluorescence intensity of the samples was obtained from HEPES buffer.
The results are shown in Table 12:
table 12: effect of different compositions on AGE formation in different tissues of mice (mean ± s, n=10)
The following is noted: p <0.01, P <0.001 compared to comparative example 1; compared to the comparative example 2 group, #P <0.01, #P <0.001
The experimental results are shown in the table. Lower AGE fluorescence values represent better anti-glycation effects. Compared with the model group, the generation of AGE in each organ of the diabetic mice is significantly inhibited to different degrees in vivo in the example 1, the example 2 and the comparative example 1, wherein the inhibition effect of the example 1 on AGE in the liver, the heart and the kidney is strongest, and the example 1 and the example 2 are also proved to have excellent anti-glycation effects.
13 Skin care effect test
40 skin-healthy subjects were selected and divided into 5 groups, and skin care products with the same function were not used for the previous week of the test.
After the face of the subject was cleaned, the skin care product was not rubbed on the face of the subject, and after waiting for 30 minutes, the skin of the face of the subject was tested for elasticity and collagen fiber level, and test data were recorded. The melissa extract prepared in each of example 1, example 2, comparative example 1 and comparative example 2 was prepared into a cream. The components of the cream are as follows:
The creams of example 1, example 2, comparative example 1 and comparative example 2 were different in that the above-described Melissa officinalis extracts prepared in example 1, example 2, comparative example 1 and comparative example 2 were used, respectively.
The creams of the example 1 group, the example 2 group, the comparative example 1 group and the comparative example 2 group were applied to the face of the subject once in the morning and at night, respectively, for 90 consecutive days, and the blank group was applied with equal amounts of distilled water. Skin elasticity and collagen fiber levels of the subject's face were tested on day 1, day 7, day 14, and day 28, respectively, and test data were recorded.
The instrument used in this experiment was a skin analysis system model CBS-1800U manufactured by Taiwan CBS.
TABLE 13
TABLE 14
The test result shows that after the skin care product containing the plant extract is used for 30 days, the skin elasticity increase rate is more than 15 percent, and the collagen fiber level is obviously improved.
The face of the subject was smeared with deionized water solutions containing the melissa extract of example 2 at different concentrations and facial creams containing the melissa extract of example 2 at different concentrations, once a day in the morning and at night, for 90 consecutive days, and the blank group was smeared with equal amounts of distilled water. Skin elasticity and collagen fiber levels of the subject's face were tested on day 1, day 7, day 14, and day 28, respectively, and test data were recorded. The components in the cream are as in example 2, and when the content of melissa officinalis therein is different, the content of deionized water therein is adjusted.
The results are shown in Table 15.
TABLE 15
14 Test of fibronectin to promote epidermal dermal layer attachment
SPF class of 3 month old adult healthy Wistar rats 160, according to reference [ Hao Chunguang, wang Lingfeng, fu Xue, etc. ] treatment effect of the selective decellularized sheepskin biological dressing on deep II degree burn rat wound [ J/CD ]. J.China injury and repair (electronic edition), 2012,7 (5): 476-481.] method was used to make 4% total surface area (total body surfacearea, TBSA) deep II degree burn models, randomly divided into iodophor gauze groups, example 1 gauze group, example 2 gauze comparative example 1 gauze group, comparative example 2 gauze group, 32 each. Example 1 gauze set was gauze soaked in 70% ethanol solution containing 0.5% of the melissa extract of example 1; example 2 gauze set was gauze soaked in 70% ethanol solution containing 0.5% of the extract of melissa officinalis of example 2; comparative example 1 is a gauze impregnated with a 70% ethanol solution containing 0.5% of the extract of melissa officinalis of comparative example 1; comparative example 2 is a gauze soaked in 70% ethanol solution containing 0.5% of the extract of Melissa officinalis of comparative example 2; the replacement is carried out at regular time every day.
And (3) sewing and fixing the peripheral No. 4-0 threads, placing two pieces of sterile gauze soaked with 0.9% sodium chloride solution on a biological dressing or an inner dressing, packaging, protecting by using a wire gauze, injecting 5mL of compound sodium chloride injection into the abdominal cavity of a rat after operation, preserving heat for resuscitation, feeding with free water after awakening, and feeding with a single cage.
RT-qPCR (reverse transcription-quantitative polymerase chain reaction) detection of mRNA (messenger ribonucleic acid) expression of LN (LN) in wound surface tissue
Each group was sacrificed at each phase point corresponding to 3, 7, 14, 28d post-injury, and the surgical area tissue was excised and stored in liquid nitrogen. About 0.3g of tissue was removed from the liquid nitrogen, placed in a pre-chilled mortar, and the tissue was ground with a pestle, with continuous addition of liquid nitrogen until ground to a powder (no visible particles apparent). TRIZOL reagent extracts total RNA from tissue. Preparing a reaction system, and sequentially carrying out reverse transcription and amplification reaction. The reverse transcription conditions were: 37 ℃ for 15min;85 ℃ for 15min; and (4) placing the reaction product in a refrigerator at the temperature of minus 20 ℃ for standby. The amplification reaction was performed using a fluorescent quantitative PCR apparatus. Each sample was provided with 5 duplicate wells. Each reaction was positive control with 28d post-injury samples and negative control with RNase-free water instead of template. Melting curve analysis procedures can show whether the PCR product is the correct amplification product or whether primer dimer is present. Primer design was performed by Primer 5.0 program according to standard fluorescent quantitative PCR Primer design principles.
TABLE 16RT-qPCR detection of laminin LN mRNA expression in burn rats
Test results show that the expression of LN mRNA is obviously improved when the gauze containing the Melissa officinalis extract is used for treatment, compared with the iodine-attached gauze group at 14d and 28d, which proves that the Melissa officinalis extract has good effect of promoting dermal epidermis link
The gauze containing the melissa extract of different concentrations prepared above and gauze containing the face cream of different concentrations were treated with gauze soaked in 70% ethanol solution containing the melissa extract of example 2 of different concentrations, otherwise consistent with the above method.
The results are shown in Table 17.
TABLE 17
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While the application has been described with reference to certain preferred embodiments, it will be understood by those skilled in the art that various changes and substitutions of equivalents may be made and equivalents will be apparent to those skilled in the art without departing from the scope of the application. Therefore, the protection scope of the application is subject to the protection scope of the claims.
Claims (12)
1. A process for preparing a melissa extract, comprising:
crushing, namely crushing the raw materials of the melissa officinalis to obtain crushed melissa officinalis;
the extraction step comprises extracting the crushed herba Melissae axillaris with a mixed solution of alcohol and water to obtain a crude extract;
filtering, namely filtering the crude extract by adopting a filter membrane to obtain filtrate;
concentrating, namely vacuum concentrating the filtrate to remove partial alcohol and water to obtain crude extract;
The resin purification step comprises loading the crude extract into an adsorption resin column, and sequentially performing water elution and gradient analysis of an alcohol-water solution to obtain an eluent;
a decoloring step, which comprises the steps of adsorbing and decoloring eluent by using active carbon and filtering to obtain decoloring liquid;
concentrating and drying, namely concentrating and drying the decolorized solution to obtain a powdery bee grass extract.
2. The method according to claim 1, wherein the mass fraction of the alcohol in the mixed solution of the alcohol and water is 50 to 80%; and/or the number of the groups of groups,
the alcohol comprises one or more of ethanol, ethylene glycol, glycerol, 1, 2-butanediol.
3. The method according to claim 2, wherein at least one of the following process conditions is fulfilled in the extracting step:
1) The ratio of the crushed materials of the melissa officinalis to the mixed solution is 1: (10-30);
2) The extraction temperature is 40-90 ℃;
3) The extraction time is 2-10 h;
4) The number of extractions is at least one;
5) At least one of stirring extraction, solvent extraction and ultrasonic extraction is adopted for extraction.
4. The method according to claim 1, wherein the filtering step comprises filtering the crude extract with a filter membrane under the conditions of negative pressure suction filtration or positive pressure filtration to obtain a filtrate; and/or the number of the groups of groups,
The pore diameter of the filter membrane is 0.22-10 mu m.
5. The method according to claim 1, wherein the mass content of ethanol in the crude extract is less than or equal to 10%.
6. The method of claim 5, wherein the adsorption resin comprises at least one of D101 type macroporous adsorption resin, AB-8 type macroporous adsorption resin, HPD100 type macroporous adsorption resin; and/or the number of the groups of groups,
the water consumption of the water elution is 3 BV-5 BV; and/or the number of the groups of groups,
the gradient analysis of the alcohol-water solution comprises the following steps: eluting with alcohol water solution of 20-30% mass fraction at 1.5-2BV/h, eluting with alcohol water solution of 4-5BV, receiving the first eluent, eluting with alcohol water solution of 40-50% mass fraction at 2-3 BV, discarding the eluent, eluting with alcohol water solution of 80-90% mass fraction at 4-5BV, receiving the second eluent, and mixing the first eluent and the second eluent to obtain the eluent.
7. The method of claim 1, wherein the decolorizing step satisfies at least one of the following process conditions:
the addition amount of the activated carbon accounts for 1-5% of the mass of the eluent;
the decoloring time is 20-60 min;
the decoloring temperature is 30-70 ℃.
8. The method of claim 1, wherein the concentrating and drying steps satisfy any one of the following process conditions:
1) Drying by using a spray dryer;
optionally, the inlet temperature of the spray dryer is 100-160 ℃, and the outlet temperature is 60-90 ℃;
optionally, the feeding speed of the concentrated decolorized solution in a spray dryer is 5-40 mL/min;
optionally, drying the concentrated decolorized solution by using a spray dryer under the condition that the compressed air pressure is 0.4-0.8 MPa;
2) The drying is carried out using a granulator, optionally at a drying temperature of 105 to 125 ℃.
9. A melissa extract characterized by being obtained by the preparation method of any one of claims 1 to 9;
optionally, the herba Melissae axillaris extract comprises flavonoids, rosmarinic acid, and caffeic acid;
optionally, the mass content of total flavonoids in the melissa officinalis extract is 40% -50%, the mass content of luteolin is 5% -10%, the mass content of rosmarinic acid is 20% -25%, and the mass content of caffeic acid is 2% -3%;
optionally, the coumarin content in the Melissa officinalis extract is below the detection limit, and further optionally < 4 μg/ml.
10. The melissa extract of claim 9, wherein melissa extract has at least one of sun protection, anti-glycation, collagen increase promotion, dermis epidermis connection promotion, and soothing.
11. Cosmetic composition comprising a melissa extract obtained by the preparation method according to any one of claims 1 to 9 or a melissa extract according to claim 10 or 11.
12. The cosmetic composition according to claim 11, wherein the mass fraction of the melissa extract in the cosmetic composition is 0.05% -10%.
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| Application Number | Priority Date | Filing Date | Title |
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| CN202310844014.0A CN116687824A (en) | 2023-07-10 | 2023-07-10 | Method for preparing herba Melissae axillaris extract, and cosmetic composition |
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| CN202310844014.0A CN116687824A (en) | 2023-07-10 | 2023-07-10 | Method for preparing herba Melissae axillaris extract, and cosmetic composition |
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