Preparation method of safflower oil-soluble extract and soothing cream using the same

Abstract

The present invention relates to a preparation method of an oil-soluble extract of safflower, belongs to the field of plant fat extraction, discloses a low-molecular amphiphilic extract, utilizes the negatively charged groups released by short-chain molecular clusters and the positively charged groups released by lysine at low temperature, quickly captures oil molecules in this dynamic equilibrium, and finally obtains an oil-soluble extract by solvent extraction, wherein the oil-soluble extract is rich in crocetin and safranal. The oil-soluble extract is emulsified into oil particles encapsulated in the middle of the liposome bilayer by modification, and modified chitosan is added to protect the structure of crocetin and nanoliposomes. Under the multi-layer protection of short-chain molecular clusters and chitosan liposome bilayers, its activity is longer-lasting, and it can be stably dispersed in a medium. It is used in a soothing cream and has the effects of relaxing the meridians and activating the collaterals, promoting blood circulation, anti-oxidation, and moisturizing.

Description

Technical Field

[0001] The invention relates to the field of plant fat extraction, in particular to a method for preparing an oil-soluble extract of safflower. Background Art

[0002] Safflower, also known as crocus, is a plant with abundant petals containing saffron essential oil. Its main components are crocetin, crocetin, and crocinaldehyde, which have been found to have antioxidant, anti-inflammatory, and antidepressant properties. Saffron inhibits oxygen free radicals, achieving its antioxidant effects. Saffron also has the benefits of promoting blood circulation and removing blood stasis, unblocking meridians, cooling blood and detoxifying, relieving inflammation and pain, enhancing immunity, detoxifying and beautifying the skin, promoting sleep, and boosting energy.

[0003] Although crocetin, crocetin, and crocinaldehyde possess numerous biological activities, they are poorly water-soluble and are fat-soluble compounds. These compounds are susceptible to decomposition reactions under light and heat conditions, which severely limit the activity of saffron oil-soluble extracts. Therefore, developing a safe, reliable, and highly active oil-soluble safflower extract is a pressing issue.

[0004] Currently, common methods for extracting oil-soluble extracts include steam distillation, organic solvent extraction, and supercritical extraction. Steam distillation involves extracting the active ingredients from plants through steam distillation and separating them to obtain an oil-soluble extract. This extraction method has the advantages of being non-toxic, environmentally friendly, and requiring simple equipment and operation. However, its low extraction rate and high energy consumption are also major drawbacks. The extracted oil-soluble substances tend to lose their activity, affecting their efficacy. Organic solvent extraction utilizes the extraction effect of a volatile solvent to extract the active ingredients of the oil-soluble substances in the plant. The organic solvent is then separated through vacuum distillation to obtain an oil-soluble extract. Organic solvent extraction has the advantages of a high extraction rate, simple equipment, easy operation, and a wide extraction range, but it is prone to leaving substances such as wax and resin in the essential oil. The active ingredients of essential oils extracted with organic solvents are mainly related to the type of solvent. Studies have shown that the antibacterial properties of essential oils vary depending on the active ingredients they contain. In addition, oils or lipophilic solvents have weak penetration into plant cells. Direct extraction with oils or lipophilic solvents results in a low yield of the target ingredients.

[0005] The Chinese patent with publication number CN118126767A discloses a method for preparing an oil-soluble extract of saffron or chicory, comprising the following steps: (1) soaking the saffron or chicory raw material in water, followed by multiple freeze-thaw cycles to obtain a feed liquid; (2) adding vegetable oil to the feed liquid, stirring and extracting, separating the oil phase after centrifugal demulsification; adding the oil phase to another feed liquid, stirring and extracting again; centrifuging and demulsifying again to separate the oil phase, adding a desiccant to remove residual water, and filtering to obtain an oil-soluble extract. The present invention destroys the structure of plant cells by repeated freeze-thaw cycles, releases intracellular substances, removes obstacles to lipophilic solvents penetrating plant cells, allows oil-soluble components to be completely in contact with lipophilic solvents, and improves yield. Although this patent solves the problem of extraction rate, it does not solve the problem of the specific component crocetin extracted by the oil-soluble extract and its activity.

[0006] Therefore, it is an urgent problem to develop an oil-soluble extract of safflower that is safe and reliable, has a high extraction rate, and has high activity and content of active substances. Summary of the Invention

[0007] In order to solve the above problems, the purpose of the present invention is to propose a method for preparing an oil-soluble extract of safflower, so as to solve one or more technical problems existing in the prior art and at least provide a beneficial choice or create conditions.

[0008] Safflower contains significant amounts of crocetin, crocetin, and crocinaldehyde. These compounds possess significant antioxidant activity, but are chemically reactive and sensitive to light and heat. Excessively high temperatures can damage their structure. Therefore, to maximize the retention of active substances during extraction, the extraction process must be performed at low temperatures. However, low-temperature extraction processes often result in low extraction yields. Therefore, a low-molecular-weight amphiphilic extractant is provided to improve the extraction yield of oil-soluble extracts.

[0009] In order to solve these problems, the present invention provides a method for preparing an oil-soluble extract of safflower, specifically:

[0010] S001, take freshly picked safflower, remove the petals and styles, wash them, and vacuum dry them at 10-20°C to remove 50% of the moisture from the petals before use;

[0011] S002, adding the dried petals and styles to the low-molecular-weight amphiphilic extract, stirring at a temperature of 20-30° C. for 120-150 minutes; performing an activation and infiltration treatment, then lowering the temperature to 2-5° C., letting it rest for about 30 minutes, then raising the temperature to 20-30° C., continuing to stir for 60-80 minutes, repeating 2-3 times, and performing a wall-breaking treatment to obtain a solid-liquid mixture;

[0012] The mass ratio of the dried petals and styles to the low-molecular-weight amphiphilic extract is 1:1 to 1:4, and the pH is 5 to 6;

[0013] S003, adding a solvent to the solid-liquid mixture and stirring for extraction. After the extraction is completed, the low-boiling point solvent is evaporated at a low temperature of 5-10°C, and then the oil phase is separated after centrifugal demulsification. A desiccant is added to remove residual water, and the mixture is filtered to obtain an oil-soluble extract. The stirring extraction temperature is 30-40°C, and the stirring extraction time is 1-2 hours. The centrifugal demulsification speed is 6000-8000 rpm, and the centrifugal demulsification time is 20-30 minutes. The desiccant is one of anhydrous calcium sulfate and anhydrous sodium sulfate.

[0014] The solvent is a homogeneous solution obtained by mixing one of rapeseed oil, linseed oil or olive oil and anhydrous ethanol in a mass ratio of 5:2;

[0015] In order to more effectively extract the oil-soluble substances in safflower, a low-molecular-weight amphiphilic extract is provided. The specific preparation method of the low-molecular-weight amphiphilic extract is as follows:

[0016] S101, adding gelatin to deionized water, heating and stirring at 30-40° C. for 30-50 minutes until the gelatin is completely dissolved, to obtain a colloidal solution;

[0017] S102, continue heating to 80-90°C, and stir rapidly at a speed of 500-800 rpm, and continue the reaction for 30-50 minutes to hydrolyze the colloidal solution to obtain a low-molecular peptide solution, cool to 20-30°C, add citric acid, adjust the pH to 5-6, and continue the reaction for 30-50 minutes to intensify the hydrolysis to obtain a short-chain molecular liquid, which is recorded as a short-chain molecular cluster solution.

[0018] S103, adding lysine to the short-chain molecular cluster solution and mixing evenly, so that the short-chain molecular cluster and lysine react with each other, so that the surface of the short-chain molecular cluster has both hydrophilic groups and lipophilic groups, and finally obtaining a low-molecular-weight amphiphilic extract containing short-chain molecular substances with a molecular weight of 1000 to 2000.

[0019] As the temperature of the gelatin solution continues to rise and the stirring accelerates, its structure begins to change. The oxygen bonds between the gelatin molecular chains are broken, the peptide bonds are hydrolyzed, and a large number of active functional groups such as amino, carboxyl and hydroxyl groups in the polypeptide side chains generate free radicals to obtain low molecular chains of polypeptide bonds. At this time, citric acid is added to further intensify the hydrolysis reaction and form a short-chain molecular liquid. The extract contains negatively ionized groups released by short-chain molecular clusters and positively ionized groups released by lysine, so that the entire extract is in a dynamic equilibrium.

[0020] The mass percentage of the gelatin solution is 5-8%, and the mass ratio of gelatin to lysine is 5-8:3-5;

[0021] Although these molecular chains in the low-molecular-weight amphiphilic extract are interrupted, the free polypeptide chains contain numerous reactive functional groups, such as amino and hydroxyl groups, on their side chains. When exposed to dried petals, the extract rapidly diffuses through the cell walls into the petal's cytosol. Reactive substances, such as crocetin, crocetin, and crocinaldehyde, present in the petal's cytosol, can undergo cross-linking reactions with the reactive functional groups in the low-molecular-weight amphiphilic extract. These substances are rapidly captured by the reactive functional groups on the short-chain molecular clusters within the low-molecular-weight amphiphilic extract, continuously aggregating and cross-linking the molecular chains. This is because the oil-soluble substances in the petals form hydrogen bonds with the amino and hydroxyl groups in the low-molecular-weight amphiphilic extract, forming a network structure within the cytosol. The carboxyl group (-COOH) or the -CHO in the aldehyde group can cross-link with the amino group. The oxygen-induced electron-withdrawing effect of the carbonyl group in the carboxyl group weakens the electronegativity of the -OH oxygen in the carboxyl group, weakening its ability to bind hydrogen. Acids, alcohols, and aldehydes abundant in the oil cells also penetrate the network of cross-linked, low-molecular-weight, short-chain molecular clusters, thereby being captured. When cooled to 2-5°C, the viscosity of the low-molecular-weight, amphiphilic extract cross-linking the oil-soluble substances increases upon cooling, forming a tighter network structure. This stretches the cell walls of the petals. Repeated high and low temperature stimulation causes the cell walls to rupture, allowing the cellular fluid to leak out, thereby improving the low-temperature extraction yield of the oil-soluble substances. The cross-linking reaction of the oil-soluble substances causes the molecular weight of the clusters to continuously aggregate and increase, also affecting their water solubility. The decrease in free amino group content and the increase in conjugated molecular weight within the clusters confirm the formation of covalent bonds between the short-chain molecular clusters and the oil-soluble substances in the petals. This conjugation process significantly enhances the antioxidant properties and thermal stability of the oil-soluble extract, but reduces the surface hydrophilicity and interfacial activity of the clusters. The resulting solid-liquid mixture is then extracted with a solvent to yield a highly active and stable oil-soluble extract.

[0022] It was found that the solubility of crocetin, the key component of the oil-soluble extract, in water was very low during use, which greatly limited the absorption and utilization of crocetin. As a biologically active substance, crocetin degrades and becomes inactivated when exposed to light or heat under natural conditions. Therefore, the use of nanoliposomes as a carrier to modify the oil-soluble extract molecules of safflower can not only further isolate the oil-soluble extract of safflower from direct contact with the external environment, thereby enhancing its stability, but also, because it is prepared into a nano system, it greatly increases the dispersibility of the oil-soluble extract in water or oil, which has a huge effect on the absorption of the oil-soluble extract in the skin layer.

[0023] In order to improve the absorption and utilization of the oil-soluble extract, we modified the obtained oil-soluble extract to obtain nanoliposomes, specifically:

[0024] S201, dissolving soybean lecithin and the oil-soluble extract in 2 times the weight of anhydrous ethanol and stirring at 400 rpm for 30 to 60 minutes to obtain a co-solvent mixture; the mass ratio of soybean lecithin to the oil-soluble extract is 1:20;

[0025] S202, dissolving chitosan in a 1% glacial acetic acid solution, adding genipin, stirring at 400 rpm for 30 to 60 minutes, and incubating in the dark for 24 hours to obtain a modified chitosan solution; the mass ratio of chitosan, 1% glacial acetic acid solution, and genipin is 1:5:1;

[0026] S203: While stirring at 400 rpm, the modified chitosan solution was slowly dripped into the co-solvent mixture and allowed to react for 2 hours. The resulting mixture was transferred to a rotary evaporator under vacuum conditions at 40°C until the ethanol was completely removed. Ultrasonic treatment was performed to obtain uniform nanoliposomes, which were then extruded through a 0.2 μm filter membrane.

[0027] Finally, a soothing cream is prepared by applying nanoliposomes.

[0028] Due to hydrophobic forces, lecithin molecules spontaneously arrange themselves into bilayer spherical vesicles in water, giving them the potential to separately encapsulate both the aqueous and oil phases. Genipin, a natural crosslinker, undergoes a Schiff base reaction with chitosan. The active groups in genipin form covalent bonds with the amino groups of chitosan, creating a specific crosslinked structure. The resulting modified chitosan solution, incubated in the dark, allows the internal structure of the nanoliposomes to crosslink more oil particles and low-molecular-weight, short-chain molecular clusters. The central aqueous phase of the liposomes encapsulates the more hydrophilic, low-molecular-weight, short-chain molecular clusters, while the oil particles can be encapsulated within the liposome bilayers. Finally, the modified chitosan crosslinked to the outer layer of the active substance not only protects the structure of crocetin and the nanoliposomes but also ensures their stable dispersion in the medium.

[0029] The oil-soluble extract is extracted using a double extraction method of low-molecular-weight amphiphilic extract and solvent. This extract not only contains rich active substances such as crocetin, crocin esters and crocin aldehyde, but also contains hydrolyzed short-chain molecular clusters of gelatin. The amphiphilicity of this short-chain molecular cluster makes these active substances highly active and more stable. Nanoliposomes are then obtained by modification with soy lecithin, and the obtained nanoliposomes can be stably dispersed in oily soothing creams.

[0030] The method provided by the present invention first allows the oil-soluble substance to be modified with gelatin to form short-chain molecular clusters, through which the amphiphilic active substances are cross-linked and efficiently extracted. The substance is then modified and prepared into nanoliposomes, in which the short-chain molecular clusters and oil-soluble particles are encapsulated. The liposomes are finally broken during massage. The hydrophilic properties of the short-chain molecular clusters are utilized to make the oil-soluble extract particle balls easily spread on the skin and easily diffuse the oil particles into the dermis of the skin, thereby exerting therapeutic effects.

[0031] Compared with the prior art, the beneficial effects of this invention are:

[0032] (1) The present invention uses a low-molecular-weight amphiphilic extract to repeatedly destroy the structure of plant cells by high and low temperatures. The low-molecular-weight amphiphilic extract is immersed in safflower oil cells, and then the negatively charged groups released by the short-chain molecular clusters and the positively charged groups released by lysine are used to quickly capture oily molecules in this dynamic equilibrium, so that the oily molecules in the oil cells are cross-linked by the low-molecular-weight amphiphilic extract, and then extracted by the solvent. This method can penetrate the barriers of plant cells even when extracted at low temperatures, and cross-link the oil-soluble components by the active groups on the short-chain molecular clusters in the amphiphilic extract, thereby improving the extraction rate. In addition, the low-molecular-weight amphiphilic extract allows the obtained oily extract to carry the short-chain molecular clusters, so that the oily extract is wrapped by the short-chain molecular clusters to obtain high activity and stability. The extraction temperature of the process of the present invention is not high, which can avoid the destruction of heat-sensitive components. Compared with conventional alcohol or water extracts, the extracted active substance content is higher, especially the extracted crocetin content is high, the activity is high, and the stability is good.

[0033] (2) The present invention also provides a nanoliposome modified from an oily extract. The central aqueous phase of the liposome encapsulates a hydrophilic low-molecular-weight short-chain molecular cluster, and the oil-soluble extract is emulsified into oil particles that can be encapsulated in the middle of the liposome bilayer. In addition, the modified chitosan can protect the structure of crocetin and the nanoliposome, making them stably dispersed in the medium. Under the multi-layer protection of the short-chain molecular cluster and the chitosan liposome bilayer, its activity lasts longer. In addition, the emulsifying effect of lecithin and the hydrophilic group effect of the short-chain molecular cluster make it uniformly dispersed in both the oil phase and the water phase, and it is easily absorbed and utilized in both the oil phase and the water phase. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 , a graph showing the change of crocetin over time in Example 1 and Comparative Example 2;

[0035] Figure 2 , a curve diagram showing the changes of safranal over time in Example 1 and Comparative Example 2. DETAILED DESCRIPTION

[0036] Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiments are only used to illustrate the present invention and are not used in limiting the scope of the present invention.In addition, should be understood that after reading the content of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms fall equally within the scope limited by the appended claims of the application.

[0037] Example 1

[0038] A method for preparing an oil-soluble extract of safflower comprises: first preparing a low-molecular-weight amphiphilic extract, specifically comprising: adding 8 parts of gelatin to 92 parts of deionized water, heating and stirring at 40° C. for 50 minutes until the gelatin is completely dissolved, thereby obtaining an 8% colloidal solution; further heating to 90° C., rapidly stirring at 800 rpm, and reacting for 50 minutes to hydrolyze the colloidal solution to obtain a low-molecular-weight peptide solution; cooling to 20° C., adding citric acid, adjusting the pH to 5.2, and reacting for 40 minutes to obtain a short-chain molecular cluster solution; and finally adding 5 parts of lysine to the short-chain molecular cluster solution and mixing uniformly to obtain a low-molecular-weight amphiphilic extract.

[0039] Furthermore, an oil-soluble extract is extracted from safflower using a low-molecular-weight amphiphilic extract, specifically:

[0040] Freshly picked safflower is taken, petals and styles are removed from leaves and washed, and the petals are vacuum dried at 10°C to remove 50% of the moisture in the petals. 100 parts of the dried petals and styles are added to 200 parts of a low-molecular-weight amphiphilic extract, and the mixture is stirred at 20°C for 120 minutes. An activation and osmosis treatment is performed, and the temperature is then lowered to 2°C, the mixture is left to stand for about 30 minutes, and then the temperature is raised to 20°C, and the mixture is stirred for 60 minutes. This is repeated three times to perform a wall-breaking treatment to obtain a solid-liquid mixture.

[0041] The extraction solvent is a homogeneous solution obtained by mixing 100 parts of rapeseed oil and 40 parts of anhydrous ethanol. At 30°C, 100 parts of the solid-liquid mixture are added to 100 parts of the solvent and stirred for extraction. After extraction for 2 hours, the mixture is evaporated at 5°C until the ethanol is completely removed. The mixture is then centrifuged at 8000 rpm for 20 minutes to separate the oil phase. Anhydrous sodium sulfate is added to remove any remaining water, and the mixture is filtered to obtain the oil-soluble extract.

[0042] Further, the obtained oil-soluble extract was modified to obtain nanoliposomes, specifically, 5 parts of soybean lecithin and 100 parts of the oil-soluble extract were dissolved in 210 parts of anhydrous ethanol, and stirred at 400 rpm for 60 minutes to obtain a co-solvent mixture;

[0043] Dissolve 5 parts of chitosan in 25 parts of 1% glacial acetic acid solution, add 5 parts of genipin, stir at 400 rpm for 40 minutes, and incubate in the dark for 24 hours to obtain a modified chitosan solution;

[0044] Under stirring at 400 rpm, 35 parts of modified chitosan solution were slowly dripped into 315 parts of the co-solvent mixture and reacted for 2 hours; the obtained mixture was transferred to a rotary evaporator for evaporation under vacuum conditions at a temperature of 40°C until the ethanol was completely removed; uniform nanoliposomes were obtained by ultrasonic treatment and finally extruded through a 0.2 μm filter membrane.

[0045] Finally, the extract is added to the soothing cream at a mass fraction of 10%. The active substance nanoliposome in the soothing cream is modified from the oil-soluble extract of safflower. First, the oil-soluble substance is cross-linked with the amphiphilic active substance of the positively charged short-chain molecular cluster made by gelatin modification and is efficiently extracted. Then, it is modified and prepared into nanoliposomes. The liposomes are wrapped with short-chain molecular clusters and oil particles, and finally rupture during use. The hydrophilic properties of the short-chain molecular clusters and the emulsifying effect of lecithin are utilized to make the oil-soluble extract particle balls easy to spread on the skin and easily diffuse the oil particles into the dermis of the skin, thereby exerting therapeutic effects. It is also beneficial to better improve the stability of the soothing cream system. The production operation is simple and convenient, and it is easy to industrialize.

[0046] Example 2

[0047] A method for preparing an oil-soluble extract of safflower comprises: first preparing a low-molecular-weight amphiphilic extract by: adding 5 parts of gelatin to 95 parts of deionized water, heating and stirring at 40°C for 40 minutes until the gelatin is completely dissolved, thereby obtaining a 5% colloidal solution; further heating the solution to 90°C, rapidly stirring at 800 rpm, and reacting for 40 minutes to hydrolyze the colloidal solution to obtain a low-molecular-weight peptide solution; cooling the solution to 20°C, adding citric acid, adjusting the pH to 5.5, and reacting for 40 minutes to obtain a short-chain molecular cluster solution; and finally adding 5 parts of lysine to the short-chain molecular cluster solution and mixing uniformly to obtain a low-molecular-weight amphiphilic extract.

[0048] Furthermore, an oil-soluble extract is extracted from safflower using a low-molecular-weight amphiphilic extract, specifically:

[0049] Freshly picked safflower is taken, petals and styles are removed from leaves and washed, and the petals are vacuum dried at 10°C to remove 50% of the moisture in the petals. 100 parts of the dried petals and styles are added to 100 parts of a low-molecular-weight amphiphilic extract, and the mixture is stirred at 30°C for 150 minutes. An activation and osmosis treatment is performed, and the temperature is then lowered to 2°C, the mixture is left to stand for about 30 minutes, and then the temperature is raised to 30°C and stirred for 80 minutes. This is repeated three times to perform a wall-breaking treatment to obtain a solid-liquid mixture.

[0050] The extraction solvent is a homogeneous solution obtained by mixing 100 parts of olive oil and 40 parts of anhydrous ethanol. At 30°C, 100 parts of the solid-liquid mixture are added to 100 parts of the solvent and stirred for extraction. After 2 hours of extraction, the mixture is evaporated at 5°C until the ethanol is completely removed. The mixture is then centrifuged at 8000 rpm for 20 minutes to separate the oil phase. Anhydrous calcium sulfate is added to remove any remaining water, and the mixture is filtered to obtain the oil-soluble extract.

[0051] Further, the obtained oil-soluble extract was modified to obtain nanoliposomes, specifically, 5 parts of soybean lecithin and 100 parts of the oil-soluble extract were dissolved in 210 parts of anhydrous ethanol, and stirred at 400 rpm for 60 minutes to obtain a co-solvent mixture;

[0052] Dissolve 5 parts of chitosan in 25 parts of 1% glacial acetic acid solution, add 5 parts of genipin, stir at 400 rpm for 50 minutes, and incubate in the dark for 24 hours to obtain a modified chitosan solution;

[0053] Under stirring at 400 rpm, 35 parts of modified chitosan solution were slowly dripped into 315 parts of the co-solvent mixture and reacted for 2 hours; the obtained mixture was transferred to a rotary evaporator for evaporation under vacuum conditions and at a temperature of 40°C, and the ethanol was completely removed by rotary evaporation; uniform nanoliposomes were obtained by ultrasonic treatment, and finally extruded through a 0.2 μm filter membrane.

[0054] Example 3

[0055] A method for preparing an oil-soluble extract of safflower comprises: first preparing a low-molecular-weight amphiphilic extract by: adding 6 parts of gelatin to 94 parts of deionized water, heating and stirring at 40°C for 40 minutes until the gelatin is completely dissolved, thereby obtaining a 6% colloidal solution; further heating the solution to 90°C, rapidly stirring at 800 rpm, and reacting for 50 minutes to hydrolyze the colloidal solution to obtain a low-molecular-weight peptide solution; cooling the solution to 30°C, adding citric acid, adjusting the pH to 5.5, and reacting for 50 minutes to obtain a short-chain molecular cluster solution; and finally adding 3 parts of lysine to the short-chain molecular cluster solution and mixing uniformly to obtain a low-molecular-weight amphiphilic extract.

[0056] Furthermore, an oil-soluble extract is extracted from safflower using a low-molecular-weight amphiphilic extract, specifically:

[0057] Freshly picked safflower is taken, petals and styles are removed from leaves and washed, and the petals are vacuum dried at 10°C to remove 50% of the moisture. 150 parts of a low-molecular-weight amphiphilic extract are added to 100 parts of the dried petals and styles, and the mixture is stirred at 30°C for 140 minutes. An activation and osmosis treatment is performed, and the temperature is then lowered to 5°C, left to stand for 30 minutes, and then raised to 30°C and stirred for 80 minutes. This is repeated three times for a wall-breaking treatment to obtain a solid-liquid mixture.

[0058] The extraction solvent is a homogeneous solution obtained by mixing 100 parts of linseed oil and 40 parts of anhydrous ethanol. At 40°C, 100 parts of the solid-liquid mixture are added to 100 parts of the solvent and stirred for extraction. After 2 hours of extraction, the mixture is evaporated at 5°C until the ethanol is completely removed. The mixture is then centrifuged at 7000 rpm for 20 minutes to separate the oil phase. Anhydrous sodium sulfate is added to remove any remaining water, and the mixture is filtered to obtain the oil-soluble extract.

[0059] Further, the obtained oil-soluble extract was modified to obtain nanoliposomes, specifically, 5 parts of soybean lecithin and 100 parts of the oil-soluble extract were dissolved in 210 parts of anhydrous ethanol, and stirred at 400 rpm for 60 minutes to obtain a co-solvent mixture;

[0060] Dissolve 5 parts of chitosan in 25 parts of 1% glacial acetic acid solution, add 5 parts of genipin, stir at 400 rpm for 60 minutes, and incubate in the dark for 24 hours to obtain a modified chitosan solution;

[0061] Under stirring at 400 rpm, 35 parts of the modified chitosan solution were slowly added dropwise to 315 parts of the co-solvent mixture and allowed to react for 2 hours. The resulting mixture was transferred to a rotary evaporator under vacuum conditions and a temperature of 40°C, and the evaporation was complete until the ethanol was completely removed. Uniform nanoliposomes were obtained by ultrasonic treatment and finally extruded through a 0.2 μm filter membrane. Comparative Example 1: When extracting an oil-soluble extract from safflower, only solvent extraction was used without adding a low-molecular-weight amphiphilic extractant. All other procedures were the same as in Example 1.

[0062] Comparative Example 2: The oil-soluble extract obtained by extraction was not modified and was directly added to prepare a soothing cream, and the other conditions were the same as those in Example 1;

[0063] Comparative Example 3: When extracting the oil-soluble extract from safflower, no low-molecular-weight amphiphilic extract was added, and only solvent extraction was used. The oil-soluble extract obtained was not modified and was directly added to the soothing cream. Other conditions were the same as in Example 1.

[0064] In Comparative Example 4, when extracting the oil-soluble extract from safflower, no low-molecular-weight amphiphilic extract was added, and the oil-soluble extract was obtained by steam distillation alone. The extracted oil-soluble extract was not modified and was directly added to the soothing cream. Other conditions were the same as in Example 1.

[0065] Test data and result analysis

[0066] 1. Main component analysis

[0067] The composition and content of the oil-soluble extracts obtained in Example 1 and Comparative Examples 1 and 4 were analyzed using a gas chromatography-mass spectrometer. Gas chromatography conditions: TG-5MS elastic quartz capillary column (0.25 mm × 30 mm; film thickness = 0.25 μm); carrier gas: high-purity helium (99.999%), carrier gas flow rate: 1.0 mL / min. Splitless injection; sample volume: 1 μL. Heating process: Initial temperature: 80°C (maintained for 2 min), heating to 200°C at a rate of 15°C / min, then heating to 280°C at a rate of 20°C / min. Maintain temperature for 15 min, inlet temperature: 250°C. Mass spectrometry conditions: MSD ion source: EI source, ion source temperature: 200°C, electron energy: 70 eV; scan range: 30-500 m / z. The NIST 05 mass spectrometry standard library was used to determine the components, and the relative content of each component was calculated using the area normalization method. See Table 1 for details.

[0068] Table 1 Content of components of oil-soluble extract

[0069]

[0070]

[0071] As can be seen from Table 1, the oil-soluble extract obtained by extracting with the solvent method of Comparative Example 1 and the steam method of Comparative Example 4 contains crocinaldehyde, but the crocetin contained is seldom or substantially not, this is because the crocetin activity is relatively high, is easy to decompose and transform into other substances during extraction, reduces the extraction amount of crocetin. And crocetin is a kind of powerful antioxidant, can effectively remove the free radicals in the body, can promote the regeneration of neurons, can alleviate anxiety and depression symptoms, enhance the immunity of the body. As can be seen from the data of Table 1, three kinds of different extraction methods, when the active substance components extracted are different, content is also very different. Especially the oil-soluble extract obtained by the steam distillation method of Comparative Example 4, this is because the temperature of distillation method is high, and extraction time is long, so that the various active contents are all different in degree destroyed, other active substances generated, its antioxidant capacity and antibacterial ability are all changed. Research has shown that, in addition to crocetin and safranal, 4,2,1,3-dihydro-3,5-dihydroxy-6-methyl-4-hydro-pyran-4-one (DDMP), octacosanol, dodecanoic acid, and 2(5H)-furanone also possess strong biological activity. Hexadecanoic acid also possesses antioxidant and antimicrobial properties, while DDMP is a strong antioxidant, while dodecanoic acid and 2(5H)-furanone possess antibacterial and antifungal properties. Therefore, safflower oil-soluble extracts not only possess superior antioxidant properties, but can also alleviate cellular aging, relieve fatigue, and strengthen the immune system. This also reveals the potential of saffron petal oil-soluble extracts as a new natural antimicrobial agent.

[0072] 2. Antibacterial performance analysis

[0073] Buffer solution: 5 g / L NH4Cl (0.088 M), 2.5 g / L NaCl (0.043 M), 15 g / L KH2PO4 (0.1 M), and 64 g / L Na2HPO4·H2O (0.24 M) were mixed evenly, diluted five times with deionized water, and adjusted to pH 7.2 for later use.

[0074] Physiological saline: Accurately weigh 9g of NaCl and completely dissolve it in 1000mL of deionized water. Seal all the above solutions and sterilize them in an autoclave at 121°C for 30 minutes before use.

[0075] LB medium: Accurately weigh 50g of LB powder and dissolve it in 2L of deionized water. Stir thoroughly to prepare LB liquid medium. Dispense the liquid medium into test tubes in 5ml portions for later use. Dispense the liquid medium into volumetric flasks in 100ml portions and add 1.3g of agar to prepare solid LB medium for later use.

[0076] PDA medium: Wash, peel, and dice potatoes. Weigh 400 g of potato chunks and add them to 2 L of deionized water. Boil on a heating mantle for 30 minutes, then filter the filtrate through gauze. Add 40 g of glucose to the filtrate and dilute to 2 L with deionized water. Once the glucose is completely dissolved, dispense the mixture into 100 mL volumetric flasks and add 1.3 g of agar to each flask to prepare solid PDA medium.

[0077] Preparation of bacterial suspension

[0078] In a cleanroom, use a sterilized inoculating loop to pick activated bacteria from the solid culture medium and place them into the previously prepared LB liquid medium. Then, mix the liquid medium by pipetting and incubate it in a 37°C shaker at 200 rpm for 12 hours. After incubation, compare the concentration of the suspension to a McFaddener turbidimeter tube and ensure that the concentration is between 10⁶ and 10⁷ CFU / mL before use. For fungi, use a two-fold dilution method to dilute the suspension to the appropriate concentration before use.

[0079] The inhibition zone experiment uses Escherichia coli, Staphylococcus aureus and Candida albicans as test bacteria, and the agar diffusion paper method is used to determine the antibacterial ability of the sample. First, put the prepared solid culture medium into a microwave oven to heat and melt. When the temperature of the culture medium drops to 50°C, add 100ul of bacterial suspension to it, mix evenly, and then slowly pour it into the culture dish. Immerse the filter paper (d=6mm) completely in the oil-soluble extract to be tested so that the oil-soluble substance is evenly distributed on the filter paper. Stick the filter paper on the previously solidified culture dish, and stick three filter paper pieces on each dish for parallel experiments. Put the air-dried culture dish upside down in a constant temperature incubator and culture it for a certain period of time (24 hours for bacterial culture and 48 hours for fungal culture), then take out the observation results and measure the diameter of the inhibition zone. Observe the sterilization situation, measure the diameter of the inhibition zone produced by each strain, measure each inhibition zone 3 times, repeat 3 times for each concentration, and take the average value. The results are shown in Table 2.

[0080] The nanoliposomes prepared in Examples 1-3 and Comparative Example 1, and the oil-soluble extracts prepared in Comparative Examples 2-4, were placed at 50°C for 30 days and then tested again for the bactericidal effects of the three bacterial species, as shown in Table 3.

[0081] Table 2 Bactericidal effect on different strains (diameter of inhibition zone, mm)

[0082] strains Example 1 Example 2 Example 3 Comparative Example 1 Comparative Example 2 Comparative Example 3 Comparative Example 4 Escherichia coli 16.5 16.1 16.6 11.3 14.8 11.3 11.0 Staphylococcus aureus 14.8 14.5 14.3 12.0 13.6 11.7 12.5 Candida albicans 18.1 17.7 18.2 8.9 13.5 9.0 8.5

[0083] Table 3 Bactericidal effect on different strains after 30 days of high temperature storage (diameter of inhibition zone, mm)

[0084]

[0085]

[0086] As shown in Table 2, the oil-soluble extracts prepared in Examples 1-3 exhibited strong inhibitory effects against Escherichia coli, Staphylococcus aureus, and Candida albicans. This may be due to the synergistic effect of the active ingredients extracted in Examples 1-3. In contrast, in Comparative Examples 1, 3, and 4, the extraction solvents used were conventional solvents, which resulted in changes in the active ingredients in the extracts. Therefore, the oil-soluble extracts prepared in these three examples had essentially no inhibitory effect on Candida albicans.

[0087] As can be seen from the antibacterial effects of Tables 2 and 3, after the oil-soluble extract is modified and prepared into nanoliposomes, its antibacterial effect does not change much, and after 30 days of high-temperature storage, it still maintains the bactericidal and antibacterial effects. However, since the oil-soluble extracts of Comparative Examples 1, 3, and 4 were not modified, their bactericidal and antibacterial effects on Escherichia coli and Staphylococcus aureus also decreased significantly after high-temperature storage. In Comparative Example 2, since the oil-soluble extract was extracted using a low-molecular-weight amphiphilic extract, the resulting oil-soluble extract had a good inhibitory effect on the three fungi. However, since the oil-soluble extract was not modified, its bactericidal and antibacterial effects also decreased significantly after high-temperature storage. Therefore, the oil-soluble extract obtained by extraction with the low-molecular-weight amphiphilic extract provided by the present invention has the best bactericidal and antibacterial effect, and the nanoliposomes obtained by modification still maintain the bactericidal and antibacterial effects after high-temperature storage.

[0088] 3. Storage stability

[0089] The nanoliposomes obtained in Example 1 and Comparative Example 2 were added at 10% by weight to prepare a soothing cream, which was heated at 50°C and stored in a blower box with a humidity of 50% for 3 months. The changes in the active ingredients crocetin and crocinaldehyde were measured by gas chromatography-mass spectrometry. Figure 1 and Figure 2 .from Figure 1 and Figure 2 It can be seen that Example 1 can be stored at high temperature for 3 months and still retains a high level of active ingredients. Figure 1-2 It can be seen that after 90 days of high-temperature storage, 85% of crocetin and 90% of safranal still remain in the soothing cream. This is because the central aqueous phase of the liposome encapsulates the hydrophilic low-molecular-weight short-chain molecular clusters, and the oil-soluble extract is emulsified into oil particles that can be encapsulated in the middle of the liposome bilayer. In addition, the modified chitosan can protect the structure of crocetin and nanoliposomes. Under the multi-layer protection of the short-chain molecular clusters and the chitosan liposome bilayer, its activity can survive longer in a high-temperature storage environment. However, in Comparative Example 2, because it does not protect the active ingredients of the oil-soluble extract, it is stored at high temperatures, resulting in the active substances being easily oxidized and deteriorating. Figure 1 Crocetin is more sensitive to high temperatures and is more likely to deteriorate at high temperatures. Therefore, if it is stored at high temperatures for 30 days without protection, it will be consumed. Therefore, it can be seen that the solution provided by the present invention can make the extracted crocetin more stable and its activity last longer.

[0090] 4. Antioxidant test

[0091] DPPH-free radical scavenging experiment

[0092] According to the "Cosmetics - Free Radical (DPPH) Scavenging Experimental Method (T / SHRH006-2018)", the DPPH free radical scavenging rate (%) of the nanoliposomes of Examples 1-3, the oil-soluble extracts obtained in Comparative Examples 1-4, and the blank control group was respectively tested.

[0093] Experimental Method: A 96-well plate was used, with three replicate wells per group, for a total of 200 μL. Sample group: Disperse an appropriate amount of the oil-soluble extract in 100 μL of distilled water to a final concentration of 5%. Add 100 μL of 0.1 mM DPPH solution to the reaction system. Control group: Add 100 μL of 0.1 mM DPPH solution to 100 μL of distilled water. After the reaction system is established, shake in the dark for 10 minutes and measure absorbance at 520 nm using a microplate reader.

[0094] The scavenging rate was calculated as follows: Scavenging rate (%) = [(A0 - Ax) / A0] × 100%, where A0 is the absorbance of the control group and Ax is the absorbance of the sample group. A higher scavenging rate indicates a more effective fermentation broth. The results of the DPPH free radical scavenging test are shown in Table 4.

[0095] Hydroxyl radical scavenging assay

[0096] Hydroxyl radicals were produced by electrocatalytic oxidation. 1g of the nanoliposomes from Examples 1-3 and the extracts from Comparative Examples 1-4 was immersed in a hydroxyl radical solution and injected into a quartz capillary tube for approximately 10 minutes for ESR (electron spin resonance) detection. The test results are shown in Table 4.

[0097] Table 4 Antioxidant test results

[0098] project Hydroxyl radical scavenging rate DPPH clearance rate Example 1 95.4％ 87.5％ Example 2 96.2％ 87.3％ Example 3 95.8％ 88.1％ Comparative Example 1 82.5％ 54.3％ Comparative Example 2 74.9％ 68.7％ Comparative Example 3 65.2％ 50.1％ Comparative Example 4 60.9％ 43.5％

[0099] As shown in Table 4, the nanoliposomes of the embodiments of the present invention have a strong scavenging ability against hydroxyl radicals and DPPH (diphenyl picrylhydrazyl radicals). Comparative Examples 1, 3, and 4 are extracted by conventional methods, and their crocetin content is very low. Moreover, they are used in an oil phase system. Since they are not modified, their active substances have weak dispersibility in the medium, which leads to a sharp decrease in antioxidant capacity. Although Comparative Example 2 uses low-molecular-weight extraction because it uses a low-molecular-weight amphiphilic extract, it is not dispersed in the oil phase and is easily oxidized, resulting in a significant decrease in antioxidant effect. Therefore, if you want the oil-soluble extract of safflower to play an antioxidant effect, on the one hand, a special solvent should be used so that the strong antioxidant crocetin can be extracted to the greatest extent, and on the other hand, crocetin should be made to have excellent dispersibility and stability in the medium. Therefore, the low-temperature active extraction method provided by the present invention first utilizes a low-molecular amphiphilic extract to be immersed in safflower oil cells, and then utilizes the negatively ionized groups released by short-chain molecular clusters and the positively ionized groups released by lysine to quickly capture oily molecules in this dynamic equilibrium, so that the oily molecules in the oil cells are cross-linked by the low-molecular amphiphilic extract and then extracted again by a solvent. Finally, the hydrophilicity of the short-chain molecular clusters and the emulsifying effect of lecithin are utilized to make the oil-soluble extract particle balls easily spread on the skin, and easily diffuse the oil particles into the dermis of the skin, thereby exerting a therapeutic effect.

[0100] The above description is only used to illustrate the technical solution of the present invention and is not intended to limit it. Any modifications and changes made to the technical solution of the present invention by ordinary persons in the art shall still fall within the scope of the present invention as long as they do not depart from the overall concept of the present invention.

Claims

1. A method for preparing an oil-soluble extract of safflower, characterized in that: The first step is to extract an oil-soluble extract from safflower, specifically: S001, take freshly picked safflower, remove the petals and styles, and wash them, then vacuum dry them at 10-20°C to remove 50% of the moisture from the petals, and set aside; S002, adding the dried petals and styles to a low-molecular-weight amphiphilic extract, stirring at a temperature of 20-30° C. for 120-150 minutes; performing an activation and infiltration treatment, then lowering the temperature to 2-5° C., letting it rest for 30 minutes, then raising the temperature to 20-30° C., continuing to stir for 60-80 minutes, repeating 2-3 times, and performing a wall-breaking treatment to obtain a solid-liquid mixture; wherein the low-molecular-weight amphiphilic extract is specifically prepared by: S101, adding gelatin to deionized water, heating and stirring at 30-40° C. for 30-50 minutes until the gelatin is completely dissolved, to obtain a colloidal solution; S102, continue heating to 80-90°C, and stir rapidly at a speed of 500-800 rpm, and continue the reaction for 30-50 minutes to hydrolyze the colloidal solution to obtain a low-molecular peptide solution, cool to 20-30°C, add citric acid, adjust the pH to 5-6, and continue the reaction for 30-50 minutes to intensify the hydrolysis to obtain a short-chain molecular liquid, which is recorded as a short-chain molecular cluster solution. S103, adding lysine to the short-chain molecular cluster solution and mixing them evenly, so that the short-chain molecular clusters and lysine react with each other, so that the short-chain molecular clusters have both hydrophilic groups and lipophilic groups on their surfaces, and finally obtaining a low-molecular-weight amphiphilic extract containing short-chain molecular substances with a molecular weight of 1000 to 2000; S003, adding a solvent to the solid-liquid mixture and stirring for extraction. After the extraction is completed, the low-boiling point solvent is evaporated at a low temperature of 5-10°C, and then the oil phase is separated after centrifugal demulsification. A desiccant is added to remove residual water, and the mixture is filtered to obtain an oil-soluble extract, wherein the stirring extraction temperature is 30-40°C, the stirring extraction time is 1-2 hours, the centrifugal demulsification speed is 6000-8000 rpm, and the centrifugal demulsification time is 20-30 minutes; the desiccant is one of anhydrous calcium sulfate and anhydrous sodium sulfate.

2. The method for preparing the oil-soluble extract of safflower according to claim 1, wherein: The solvent is a homogeneous solution obtained by uniformly mixing one of rapeseed oil, linseed oil or olive oil and anhydrous ethanol in a mass ratio of 5:

2.

3. The method for preparing the oil-soluble extract of safflower according to claim 1, wherein: The mass ratio of the dried petals and styles to the low-molecular-weight amphiphilic extract is 1:1-1:4, and the pH value is 5-6; the mass ratio of the solid-liquid mixture to the solvent is 1:

1.

4. The method for preparing the oil-soluble extract of safflower according to claim 1, wherein: The mass percentage of the gelatin solution is 5-8%, and the mass ratio of gelatin to lysine is 5-8:3-5.

5. The method for preparing the oil-soluble extract of safflower according to claim 1, wherein: The obtained oil-soluble extract is modified to obtain nanoliposomes, specifically: S201, dissolving soybean lecithin and the oil-soluble extract in 2 times the weight of anhydrous ethanol, and stirring at 400 rpm for 30 to 60 minutes to obtain a co-solvent mixture; the mass ratio of soybean lecithin to the oil-soluble extract is 1:20; S202, dissolving chitosan in a 1% glacial acetic acid solution, adding genipin, stirring at 400 rpm for 30 to 60 minutes, and incubating in the dark for 24 hours to obtain a modified chitosan solution; the mass ratio of chitosan, 1% glacial acetic acid solution, and genipin is 1:5:1; S203, slowly dripping the modified chitosan solution into the co-solvent mixture while stirring at 400 rpm, and reacting for 2 hours; transferring the obtained mixture to a rotary evaporator for evaporation, setting the conditions to vacuum conditions and the temperature to 40°C, and rotary evaporating until the ethanol is completely removed; obtaining uniform nanoliposomes by ultrasonic treatment, and finally extruding through a 0.2 μm filter membrane.

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