Preparation method of oil-soluble extract of safflower carthamus and soothing cream applying oil-soluble extract of safflower carthamus

Abstract

The invention relates to a preparation method of an oil-soluble extract of safflower carthamus, belongs to the field of plant fat extraction, and discloses a low-molecular amphiphilic leaching solution which is used for rapidly capturing oil molecules in dynamic balance by utilizing groups with negative ions released by short-chain molecular clusters and groups with positive ions released by lysine at low temperature, so that the oil-soluble extract of safflower carthamus is obtained. Finally, the oil-soluble extract is obtained through solvent extraction, and the oil-soluble extract is rich in crocetin and crocetin. An oil-soluble extract is emulsified into oil particles through modification to be encapsulated in the middle of a liposome bimolecular layer, and modified chitosan can protect structures of crocetin and the nano-liposome, so that under multi-layer protection of the short-chain molecular cluster and the chitosan liposome bimolecular layer, the crocetin nano-liposome has longer activity, can be stably dispersed in a medium, and has good stability. When being used in soothing cream, the composition has the effects of soothing channels and activating collaterals, promoting blood circulation, resisting oxidation and moistening.

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 saffron, crocus. Its petals are rich in a large amount of saffron petal essential oil. At present, saffron essential oil has been found to have active substances such as crocetin, crocin esters and crocin aldehyde, which have anti-oxidation, anti-inflammatory, anti-depression and other pharmacological effects. Saffron has an inhibitory effect on oxygen free radicals, thereby achieving an antioxidant effect; saffron also has the effects of promoting blood circulation and removing blood stasis, unblocking meridians, cooling blood and detoxifying, anti-inflammatory and analgesic, improving human immunity, detoxifying and beautifying, promoting sleep, and improving energy.

[0003] Although crocetin, crocin and crocinaldehyde have many biological activities, they are poorly water-soluble and are a class of fat-soluble compounds. They are prone to decomposition reactions and lose their activity under light and heat conditions. These properties severely limit the activity of saffron oil-soluble extracts. Therefore, developing a safe and reliable saffron oil-soluble extract with high active ingredients is one of the problems that need to be solved urgently.

[0004] At present, the common extraction methods of oil-soluble extracts include steam distillation, organic solvent extraction and supercritical extraction. Steam distillation is to take out the active ingredients in the plant through steam distillation and obtain oil-soluble extracts after separation. This extraction method has the advantages of non-toxicity, environmental protection, simple equipment and operation, but at the same time, its low extraction rate and high energy consumption have become its biggest drawbacks, and the extracted oil-soluble substances are easy to lose activity and affect the efficacy. The organic solvent extraction method uses the extraction effect of volatile solvents to extract the active ingredients of oil-soluble substances in plants, and then separates the organic solvent through reduced pressure distillation to obtain oil-soluble extracts. Organic solvent extraction has the advantages of high extraction rate, simple equipment, convenient operation and wide extraction range, but it is easy to leave substances such as wax and resin in the plant 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, oil or lipophilic solvents have weak penetration into plant cells. Direct extraction with oil 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, separating the oil phase, adding a desiccant to remove residual water, filtering, and obtaining an oil-soluble extract. The present invention destroys the structure of plant cells through repeated freeze-thaw cycles, releases intracellular substances, removes obstacles to lipophilic solvents penetrating plant cells, and allows the oil-soluble components to be completely in contact with the lipophilic solvent, thereby increasing the yield. Although the patent solves the problem of extraction rate, it does not solve the problem of the specific component crocetin extracted from the oil-soluble extract and its activity.

[0006] Therefore, it is an urgent problem to develop an oil-soluble extract of safflower which 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 a large amount of crocetin, crocin esters and crocin aldehyde, all of which have good antioxidant activity, but their chemical properties are relatively active and sensitive to light and heat. Excessive temperature will cause certain damage to its structure, so in order to retain the active substances to the greatest extent during the extraction process, it is necessary to ensure that the extraction process is carried out at a low temperature. However, in the low-temperature extraction process, there is a problem of low extraction rate, so a low-molecular amphiphilic extract is also provided to improve the extraction rate 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, which is specifically:

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

[0011] S002, adding the dried petals and styles to the low molecular weight amphiphilic extract, stirring at 20-30° C. for 120-150 minutes; performing activation and penetration treatment, then lowering the temperature to 2-5° C., letting it stand for about 30 minutes, then heating it to 20-30° C., continuing to stir for 60-80 minutes, repeating 2-3 times, performing wall breaking treatment, and obtaining 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 value is 5 to 6;

[0013] S003, add solvent to the solid-liquid mixture and stir to extract. After the extraction, evaporate the low boiling point solvent at 5-10°C, then separate the oil phase by centrifugal demulsification. Add desiccant to remove residual water, filter, and obtain oil-soluble extract, wherein the stirring extraction temperature is 30-40°C, and the stirring extraction time is 1-2 hours. The centrifugal demulsification speed is 6000-8000rpm, and the centrifugal demulsification time is 20-30min. 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 to 40° C. for 30 to 50 minutes until the gelatin is completely dissolved, to obtain a colloidal solution;

[0017] S102, continue to heat to 80-90°C, and stir rapidly at a speed of 500-800 rpm, continue to react for 30-50 minutes, hydrolyze the colloidal solution to obtain a low-molecular peptide solution, cool to 20-30°C, add citric acid, adjust the pH value to 5-6, continue to react for 30-50 minutes, intensify the hydrolysis, and 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 produce 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, there are a large number of active functional groups such as amino and hydroxyl groups on the side chains of the free polypeptide chains. When they come into contact with the dried petals, the extract quickly diffuses into the cell sap of the petals through the cell wall, and the active substances such as crocetin, crocin esters and crocin aldehyde in the cell sap of the petals can undergo cross-linking reactions with the active functional groups in the low-molecular-weight amphiphilic extract, and are quickly captured by the active functional groups on the short-chain molecular clusters in the low-molecular-weight amphiphilic extract, and continuously aggregate and cross-link on the molecular chains. This is because the oil-soluble substances in the petals and the amino and hydroxyl groups in the low-molecular-weight amphiphilic extract undergo hydrogen bonds, forming a network structure inside the cell sap, and the carboxyl -COOH or -CHO in the aldehyde group can undergo cross-linking with the amino group, and the oxygen-induced effect of the carbonyl group in the carboxyl group absorbs electrons, which weakens the electronegativity of the -OH oxygen in the carboxyl group and its ability to bind H. Acidic substances, alcohols and aldehydes rich in oil cells will also penetrate into the network of cross-linked low-molecular short-chain molecular clusters and be captured; when the temperature is reduced to 2-5°C, the viscosity of the low-molecular amphiphilic extract that cross-links oil-soluble substances increases after cooling, forming a tighter network structure, which makes the cell wall of the petals expand. Repeated high and low temperature stimulation causes the cell wall of the petals to rupture, and the substances in the cell fluid flow out, which improves the low-temperature extraction rate of oil-soluble substances. Because of the cross-linking reaction of oil-soluble substances, the molecular weight of this molecular cluster continues to aggregate and increase, and also affects its water solubility. The reduction of free amino content in the molecular cluster and the increase of conjugated molecular weight confirm that covalent bonds are formed between the short-chain molecular cluster and the oil-soluble substances in the petals. The conjugation process significantly improves the antioxidant properties and thermal stability of the oil-soluble extract, but reduces the surface hydrophilicity and interfacial activity of the molecular cluster. Finally, the obtained solid-liquid mixture is extracted with a solvent to obtain an oil-soluble extract with high activity and high stability.

[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 will degrade and become inactive when exposed to light and heat under natural conditions. Therefore, further, the use of nanoliposomes as a carrier to modify the oil-soluble extract molecules of safflower can not only further isolate the direct contact between the oil-soluble extract of safflower and the external environment to enhance 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 oil-soluble extract in 2 times the weight of anhydrous ethanol, and stirring at a speed of 400 rpm for 30 to 60 minutes to obtain a co-dissolved mixed solution; the mass ratio of soybean lecithin to oil-soluble extract is 1:20;

[0025] S202, dissolving chitosan in a 1% glacial acetic acid solution, adding genipin, stirring at a speed of 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, under stirring at 400 rpm, slowly drip the modified chitosan solution into the co-solvent mixture, react for 2 hours; transfer the obtained mixture to a rotary evaporator for evaporation, set the conditions to vacuum conditions, set the temperature to 40°C, and evaporate until the ethanol is completely removed. Obtain uniform nanoliposomes through ultrasonic treatment, and finally extrude through a 0.2 μm filter membrane.

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

[0028] Due to the hydrophobic force, lecithin molecules spontaneously arrange into a bilayer spherical vesicle structure in water, which makes it have the potential to encapsulate the water phase and the oil phase respectively. Genipin, a natural cross-linking agent, reacts with chitosan to form a Schiff base. The active groups in genipin form covalent bonds with the amino groups of chitosan, thereby forming a certain cross-linking structure. The modified chitosan solution obtained after incubation in the dark can make the internal structure of the nanoliposomes cross-link more oil particles and low-molecular short-chain molecular clusters. The central water phase of the liposome encapsulates the hydrophilic low-molecular short-chain molecular clusters, while the oil particles can be encapsulated in the middle of the bilayer of the liposome. Finally, the modified chitosan cross-linked in the outer layer of the active substance can not only protect the structure of crocetin and nanoliposomes, but also make them stably dispersed in the medium.

[0029] The oil-soluble extract is extracted by a double extraction method using a low-molecular-weight amphiphilic extractant and a solvent. The 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 these short-chain molecular clusters 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 by gelatin to form short-chain molecular clusters through cross-linking of amphiphilic active substances so as to be efficiently extracted, and then modified to be prepared into nanoliposomes, in which short-chain molecular clusters and oil-soluble particles are encapsulated, and finally ruptured 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 the oil particles are easily diffused into the dermis of the skin, thereby exerting therapeutic effects.

[0031] Compared with the prior art, the invention has the following beneficial effects:

[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 balance, so that the oily molecules in the oil cells are cross-linked by the low-molecular-weight amphiphilic extract, and then extracted by a solvent; this method realizes that low-temperature extraction can also penetrate the barriers of plant cells, and the oil-soluble components are cross-linked by the active groups on the short-chain molecular clusters in the amphiphilic extract to improve the extraction rate, and the low-molecular-weight amphiphilic extract is used to make the obtained oily extract carry 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, wherein the central aqueous phase of the liposome encapsulates a hydrophilic low-molecular 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, and 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 emulsification effect of lecithin and the hydrophilic group effect of the short-chain molecular cluster make it evenly 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 chart showing the change of safranal over time in Example 1 and Comparative Example 2. DETAILED DESCRIPTION

[0036] The present invention will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and are not intended to limit the scope of the present invention. In addition, it should be understood that after reading the content described in the present invention, those skilled in the art may make various changes or modifications to the present invention, and these equivalent forms fall within the scope limited by the appended claims of the application equally.

[0037] Example 1

[0038] A method for preparing an oil-soluble extract of safflower, comprising: first preparing a low-molecular-weight amphiphilic extract, specifically: 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, to obtain an 8% colloidal solution; continuing to heat to 90°C, rapidly stirring at 800 rpm, reacting for 50 minutes, hydrolyzing the colloidal solution to obtain a low-molecular-weight peptide solution, cooling to 20°C, adding citric acid, adjusting the pH value to 5.2, reacting for 40 minutes, to obtain a short-chain molecular cluster solution; adding 5 parts of lysine to the short-chain molecular cluster solution and mixing evenly, to finally obtain a low-molecular-weight amphiphilic extract;

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

[0040] Take freshly picked safflower, remove leaves from petals and styles, wash and dry them, perform vacuum drying at 10°C to remove 50% of the moisture in the petals, add 100 parts of the dried petals and styles into 200 parts of low-molecular-weight amphiphilic extract, stir for 120 minutes at 20°C, perform activation and penetration treatment, lower the temperature to 2°C, let stand for about 30 minutes, then raise the temperature to 20°C, continue stirring for 60 minutes, repeat for 3 times, perform wall breaking treatment, and obtain a solid-liquid mixture;

[0041] Take 100 parts of rapeseed oil and 40 parts of anhydrous ethanol and mix them evenly to obtain a homogeneous solution as the extraction solvent. At a temperature of 30°C, take 100 parts of the solid-liquid mixture and add 100 parts of the solvent to stir and extract. After extraction for 2 hours, evaporate at 5°C until the ethanol is completely removed; then centrifuge at a speed of 8000rpm for 20 minutes to separate the oil phase; add anhydrous sodium sulfate to remove the residual water, filter, and 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 a speed of 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 into 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 prepared by gelatin modification and is efficiently extracted. Then, it is modified and prepared into nanoliposome. The liposome wraps the short-chain molecular cluster and the oil particles, and finally breaks during use. The hydrophilic property of the short-chain molecular cluster and the emulsifying effect of lecithin are utilized to make the oil-soluble extract particle ball spread on the skin easily, and the oil particles are easily diffused into the dermis of the skin, so as to exert the therapeutic effect. 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 be industrialized.

[0046] Example 2

[0047] A method for preparing an oil-soluble extract of safflower, comprising: first preparing a low-molecular-weight amphiphilic extract, specifically: 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, to obtain a 5% colloidal solution; continuing to heat to 90°C, and rapidly stirring at 800 rpm, reacting for 40 minutes, hydrolyzing the colloidal solution to obtain a low-molecular-weight peptide solution, cooling to 20°C, adding citric acid, adjusting the pH value to 5.5, reacting for 40 minutes, to obtain a short-chain molecular cluster solution; adding 5 parts of lysine to the short-chain molecular cluster solution and mixing evenly, to finally obtain a low-molecular-weight amphiphilic extract;

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

[0049] Take freshly picked safflower, remove leaves from petals and styles, wash and dry them, dry them at 10°C in a vacuum dryer to remove 50% of the moisture in the petals, add 100 parts of the dried petals and styles into 100 parts of a low-molecular-weight amphiphilic extract, stir them at 30°C for 150 minutes, perform an activation and penetration treatment, lower the temperature to 2°C, let it stand for about 30 minutes, then raise the temperature to 30°C, continue to stir for 80 minutes, repeat for 3 times, perform a wall-breaking treatment, and obtain a solid-liquid mixture;

[0050] Take 100 parts of olive oil and 40 parts of anhydrous ethanol and mix them evenly to obtain a homogeneous solution as the extraction solvent. At a temperature of 30°C, take 100 parts of the solid-liquid mixture and add 100 parts of the solvent to stir and extract. After extraction for 2 hours, evaporate at 5°C until the ethanol is completely removed; then centrifuge at a speed of 8000rpm for 20 minutes to separate the oil phase; add anhydrous calcium sulfate to remove the residual water, filter, and 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 a speed of 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, the conditions were set to vacuum conditions, the temperature was set to 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, comprising: first preparing a low-molecular-weight amphiphilic extract, specifically: 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, to obtain a 6% colloidal solution; continuing to heat to 90°C, and rapidly stirring at 800 rpm, reacting for 50 minutes, hydrolyzing the colloidal solution to obtain a low-molecular-weight peptide solution, cooling to 30°C, adding citric acid, adjusting the pH value to 5.5, reacting for 50 minutes, to obtain a short-chain molecular cluster solution; adding 3 parts of lysine to the short-chain molecular cluster solution and mixing evenly, to finally obtain a low-molecular-weight amphiphilic extract;

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

[0057] Take freshly picked safflower, remove leaves from petals and styles, wash and dry them, dry them at 10°C in a vacuum oven to remove 50% of the moisture in the petals, add 150 parts of low-molecular-weight amphiphilic extract to 100 parts of the dried petals and styles, stir them at 30°C for 140 minutes, perform activation and penetration treatment, lower the temperature to 5°C, let it stand for 30 minutes, then raise the temperature to 30°C, continue stirring for 80 minutes, repeat for 3 times, perform wall breaking treatment and obtain a solid-liquid mixture;

[0058] Take 100 parts of linseed oil and 40 parts of anhydrous ethanol and mix them evenly to obtain a homogeneous solution as the extraction solvent. At a temperature of 40°C, take 100 parts of the solid-liquid mixture and add 100 parts of the solvent to stir and extract. After extraction for 2 hours, evaporate at 5°C until the ethanol is completely removed; then centrifuge at a speed of 7000rpm for 20 minutes to separate the oil phase; add anhydrous sodium sulfate to remove the residual water, filter, and 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 a speed of 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 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, the conditions were set to vacuum conditions, the temperature was set to 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. Comparative Example 1, when extracting an oil-soluble extract from safflower, no low molecular weight amphiphilic extract was added, and only solvent extraction was used, and the rest was the same as Example 1;

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

[0063] Comparative Example 3, when extracting the oil-soluble extract from safflower, no low-molecular-weight amphiphilic extract was added, only solvent extraction was used, and the oil-soluble extract obtained was not modified, but directly added to the soothing cream, and the rest was the same as in Example 1;

[0064] In Comparative Example 4, when extracting the oil-soluble extract from safflower, no low-molecular-weight amphiphilic extracting solution is added, and the oil-soluble extract is obtained by only using steam distillation, and the extracted oil-soluble extract is not modified and is directly added to the soothing cream. The rest is 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 by gas chromatography-mass spectrometry. Gas chromatography conditions: TG-5MS elastic quartz capillary column (0.25mm×30mm; film thickness = 0.25μm); carrier gas is high-purity helium (99.999%), and the carrier gas flow rate is 1.0mL / min. No split injection; sample volume 1μL. Heating process: initial temperature 80℃ (maintained for 2min), heated to 200℃ at a rate of 15℃ / min, and then heated to 280℃ at a rate of 20℃ / min. Constant temperature for 15min, inlet temperature is 250℃. Mass spectrometry conditions: MSD ion source uses EI source, ion source temperature is 200℃, electron energy is 70eV; scanning range: 30~500m / z. The NIST 05 mass spectrometry standard library is used to determine each component, and the relative content of each component is calculated by area normalization method, as shown in Table 1.

[0068] Table 1 Oil-soluble extract ingredient content

[0069]

[0070]

[0071] As can be seen from Table 1, the oil-soluble extract obtained by the solvent method of Comparative Example 1 and the water vapor method of Comparative Example 4 contains crocinaldehyde, but the crocetin contained is rarely or substantially absent, because the activity of crocetin is relatively high, and it is easy to decompose and transform into other substances during the extraction process, reducing the amount of crocetin extracted. And crocetin is a powerful antioxidant, which can effectively remove free radicals in the body, promote the regeneration of neurons, relieve anxiety and depression symptoms, and enhance the body's immunity. It can also be seen from the data of Table 1 that the three different methods of extraction, while the active substance components extracted are different, the content is also very different. Especially the oil-soluble extract obtained by the water vapor distillation method of Comparative Example 4, this is because the temperature of the distillation method is high, the extraction time is long, so that various active contents are destroyed to different degrees, and other active substances generated, its antioxidant capacity and antibacterial capacity are changed. Studies have 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 have strong biological activity. Hexadecanoic acid also has antioxidant and antibacterial properties. DDMP is a strong antioxidant, while dodecanoic acid and 2(5H)-furanone have antibacterial and antifungal properties. Therefore, the oil-soluble extract of safflower not only has excellent antioxidant capacity, but also can relieve cell aging, relieve fatigue of the body and enhance the immune system. This also reveals the potential of saffron petal oil-soluble extract as a new natural antibacterial agent.

[0072] 2. Antibacterial performance analysis

[0073] Buffer solution: Mix 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) respectively, dilute five times with deionized water, and adjust the pH to 7.2 for later use.

[0074] Physiological saline: Accurately weigh 9g of NaCl and completely dissolve it in 1000mL of deionized water for later use. All the above prepared solutions are sealed and placed in an autoclave for high temperature sterilization at 121℃ for 30min before use.

[0075] LB medium: Accurately weigh 50g LB powder and dissolve it in 2L deionized water and stir evenly to obtain LB liquid medium. The liquid medium is packed into test tubes in 5ml batches for later use. Then, the liquid medium is packed into a volumetric flask in 100ml batches and 1.3g agar is added to the flask to make a solid LB medium for later use.

[0076] PDA medium: Wash and peel potatoes and cut them into pieces. Weigh 400g of potato pieces, add them to 2L of deionized water, boil them on a heating mantle for 30 minutes, and filter out the filtrate with gauze. Add 40g of glucose to the filtrate and dilute to 2L with deionized water. After the glucose is completely dissolved, dispense it into 100ml volumetric flasks and add 1.3g of agar to each flask to make solid PDA medium.

[0077] Preparation of bacterial suspension

[0078] In the clean bench, use a sterilized inoculation loop to pick up the activated bacteria on the solid culture medium and place them in the previously prepared LB liquid culture medium. Then, place the mixed liquid culture medium in a 37°C constant temperature shaker at 200 rpm for 12 hours. After the shaker culture, compare the McFarland turbidimeter tube and prepare the bacterial suspension to ensure that its concentration is 106-107 CFU / mL for later use. For fungi, use the two-fold dilution method to dilute the bacterial suspension to an appropriate concentration for later use.

[0079] The antibacterial 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, heat the prepared solid culture medium in a microwave oven to 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 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 antibacterial zone. Observe the sterilization situation, measure the diameter of the antibacterial zone produced by each strain, measure each antibacterial 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 the bactericidal effects of the three strains were tested again, as shown in Table 3;

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

[0082] Bacteria 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 can be seen from Table 2, the oil-soluble extracts prepared in Examples 1-3 showed strong inhibitory effects on Escherichia coli, Staphylococcus aureus, and Candida albicans. This may be due to the synergistic effect of the active substances extracted in Examples 1-3. In Comparative Examples 1, 3, and 4, because the extraction solvent is a common solvent, the active ingredients in the extracted substances change, so the three oil-soluble extracts prepared have basically no inhibitory effect on Candida albicans.

[0087] From the antibacterial effects of Tables 2 and 3, it can be seen that after the oil-soluble extract is modified and prepared into nanoliposomes, its antibacterial effect does not change much, and after being stored at high temperature for 30 days, it still maintains the bactericidal and antibacterial effects. However, since the oil-soluble extracts of Comparative Examples 1, 3, and 4 are not modified, after being stored at high temperature, their bactericidal and antibacterial effects on Escherichia coli and Staphylococcus aureus also decrease significantly. Since Comparative Example 2 is extracted by using a low-molecular amphiphilic extract, the obtained oil-soluble extract has a good inhibitory effect on the three kinds of fungi, but because its oil-soluble extract is not modified, after being stored at high temperature, its bactericidal and antibacterial effect also decreases significantly. Therefore, the oil-soluble extract obtained by extracting with the low-molecular amphiphilic extract provided by the present invention has the best bactericidal and antibacterial effect, and the bactericidal and antibacterial effect of the nanoliposome obtained by modification is still maintained 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 relatively 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 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. Comparative Example 2 does not protect the active ingredients of the oil-soluble extract. It is stored at high temperatures, which makes the active substances easily oxidized and deteriorated. Figure 1 Crocetin is more afraid of high temperature and is more likely to deteriorate at high temperature. Therefore, if it is stored at high temperature 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 active for a longer time.

[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 rates (%) of the nanoliposomes of Examples 1-3, the oil-soluble extracts obtained in Comparative Examples 1-4, and the blank control group were respectively detected.

[0093] Experimental method: Use a 96-well plate, set up three replicate wells in each group, and the system is 200μL. Sample group: Take an appropriate amount of oil-soluble extract sample, disperse it in 100μL distilled water, so that the final concentration of the sample to be tested in the system is 5%, and add 100μL 0.1mM DPPH solution to the reaction system; control group: Take 100μL distilled water, and then add 100μL 0.1mM DPPH solution. After the reaction system is constructed, shake it in the dark for 10 minutes, and use an enzyme reader to test the absorbance at 520nm.

[0094] The calculation method of the clearance rate is: clearance rate (%) = [(A0-Ax) / A0] × 100%, A0 is the absorbance of the control group, and Ax is the absorbance of the sample group. The higher the clearance rate, the better the effect of the fermentation liquid. The results of the DPPH free radical scavenging test are shown in Table 4.

[0095] Hydroxyl free radical scavenging assay

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

[0097] Table 4 Antioxidant test results

[0098] project Hydroxyl radical scavenging rate DPPH clearance 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 embodiment of the present invention have strong scavenging ability against hydroxyl radicals and DPPH (diphenyl picrylhydrazyl radicals). Comparative Examples 1, 3 and 4 are extracted by ordinary methods, and the content of crocetin is very small. Moreover, they are used in the oil phase system. Because they are not modified, the active substances have weak dispersibility in the medium, which makes the antioxidant ability drop sharply. Although Comparative Example 2 uses low molecular weight extraction because it uses low molecular weight amphiphilic extract, it is not dispersed in the oil phase and is easily oxidized, so the antioxidant effect is also significantly deteriorated. Therefore, if the oil-soluble extract of safflower is desired to exert its antioxidant effect, on the one hand, a special solvent should be used to make the crocetin, a strong antioxidant, 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 low-molecular amphiphilic extracting liquid to immerse 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 balance, so that the oily molecules in the oil cells are cross-linked by the low-molecular amphiphilic extracting liquid, and then extracted by the solvent. Finally, the hydrophilicity of the short-chain molecular clusters and the emulsification of lecithin are utilized to make the oil-soluble extract particle balls easily spread on the skin, and the oil particles are easily diffused to the dermis of the skin, thereby exerting the curative effect.

[0100] The above description is only used to illustrate the technical solution of the present invention rather than to limit it. Any equal modifications and changes made to the technical solution of the present invention by ordinary persons in the art, as long as they do not deviate from the overall concept of the present invention, are still within the scope 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, remove the leaves, wash, and vacuum dry at 10-20°C to remove 50% of the moisture from the petals, and set aside; S002, adding the dried petals and styles to the low molecular weight amphiphilic extract, stirring at a temperature of 20 to 30° C. for 120 to 150 minutes; performing activation and penetration treatment, then lowering the temperature to 2 to 5° C., letting it stand for 30 minutes, then heating it to 20 to 30° C., continuing to stir for 60 to 80 minutes, repeating 2 to 3 times, performing wall breaking treatment, and obtaining a solid-liquid mixture; S003, adding solvent to the solid-liquid mixture and stirring extraction, after the extraction is completed, distilling off the low boiling point solvent at a low temperature of 5 to 10°C, and then separating the oil phase after centrifugal demulsification; adding a desiccant to remove residual water, filtering, and obtaining an oil-soluble extract, wherein the stirring extraction temperature is 30 to 40°C, the stirring extraction time is 1 to 2 hours; the centrifugal demulsification speed is 6000 to 8000 rpm, and the centrifugal demulsification time is 20 to 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, characterized in that: 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, characterized in that: 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, characterized in that: The specific preparation method of the low-molecular-weight amphiphilic extract is as follows: S101, adding gelatin to deionized water, heating and stirring at 30 to 40° C. for 30 to 50 minutes until the gelatin is completely dissolved, to obtain a colloidal solution; S102, continue to heat to 80-90°C, and stir rapidly at a speed of 500-800 rpm, continue to react for 30-50 minutes, hydrolyze the colloidal solution to obtain a low-molecular peptide solution, cool to 20-30°C, add citric acid, adjust the pH value to 5-6, continue to react for 30-50 minutes, intensify the hydrolysis, and 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 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; 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, characterized in that: The obtained oil-soluble extract is modified to obtain nanoliposomes, specifically: S201, dissolving soybean lecithin and oil-soluble extract in 2 times the weight of anhydrous ethanol, and stirring at a speed of 400 rpm for 30 to 60 minutes to obtain a co-dissolved mixed solution; the mass ratio of soybean lecithin to oil-soluble extract is 1:20; S202, dissolving chitosan in a 1% glacial acetic acid solution, adding genipin, stirring at a speed of 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 drip the modified chitosan solution into the co-solvent mixture under stirring at 400 rpm, and react for 2 hours; transfer the obtained mixture to a rotary evaporator for evaporation, set the conditions to vacuum conditions, set the temperature to 40°C, and rotary evaporate until the ethanol is completely removed; obtain uniform nanoliposomes through ultrasonic treatment, and finally extrude through a 0.2 μm filter membrane.

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