Extraction method and application of stigma-removed saffron crocus flower oil-soluble component

Abstract

The invention relates to the field of daily chemical products, in particular to an extraction method and application of stigma-removed saffron crocus oil-soluble components, and the extraction method comprises the following steps: performing enzymolysis treatment on stigma-removed saffron crocus flowers by using an enzyme preparation to obtain a first reactant; carrying out extraction and purification treatment on the first reactant by utilizing ultrasound and a deep-eutectic solvent to obtain a second reactant, and carrying out extraction treatment and centrifugal treatment on the second reactant to obtain a third reactant; and concentrating to obtain the stigma-removed saffron crocus flower oil-soluble component. According to the method, the stigma-removed saffron crocus flowers are subjected to enzymolysis by using the enzyme preparation, so that cell walls of the stigma-removed saffron crocus flowers are destroyed, release of effective components is promoted, oil-soluble components in cells are released, and the subsequent extraction efficiency is improved. And purifying a reactant after enzymolysis by utilizing ultrasonic and a deep-eutectic solvent. Ultrasonic waves can enhance permeation and dissolution of the solvent to target components, the eutectic solvent can provide a milder extraction environment, and the whole extraction method enables the extraction rate and the content of active components to be remarkably increased.

Description

Technical Field

[0001] The invention relates to the field of daily chemical products, and in particular to a method for extracting oil-soluble components of saffron without stigma and application thereof. Background Art

[0002] Saffron (Crocus sativus L), also known as saffron, is a perennial herb of the genus Crocus in the family Iridaceae. It is mainly planted in Central Asia and Europe in the world. Saffron is a traditional precious medicinal material. In modern medicine, the medicinal part of saffron, namely the stigma of saffron, has the effects of promoting blood circulation and removing blood stasis, unblocking meridians, cooling blood and detoxifying, anti-inflammatory and analgesic, and treating depression. However, only the stigma of saffron is used as medicine, and the yield is extremely low. Because its resources are extremely limited and cannot meet the actual production needs, the price is very high, and it has always been known as "plant gold".

[0003] Since the stigma of saffron only accounts for 7.4% of the weight of the whole flower, it means that about 170,000 flowers must be picked to obtain 1 kg of stigma, and the remaining flowers without stigma are often abandoned, resulting in a huge waste of resources. Therefore, the development and utilization of non-traditional medicinal parts of saffron have received attention. Studies have shown that the flowers without stigma of saffron contain flavonoids, phenolic acids, monoterpenes, glycosides, alkaloids, anthraquinones, saponins and other ingredients, which have pharmacological activities such as antioxidant, anti-inflammatory, anti-obesity and dyslipidemia. Therefore, the development of the utilization value of non-traditional medicinal parts of saffron, especially to improve the extraction rate of oil-soluble components in the flowers without stigma, has important scientific significance and application value. Summary of the invention

[0004] (I) Purpose of the invention

[0005] The invention aims to provide a method for extracting oil-soluble components from stigma-free saffron flower parts and application thereof, which can improve the extraction rate.

[0006] (II) Technical solution

[0007] In order to solve the above problems, the present invention provides a method for extracting oil-soluble components from the flower part of saffron without stigma, comprising:

[0008] Using an enzyme preparation to enzymatically hydrolyze the flower part of saffron without stigma to obtain a first reactant;

[0009] Extracting and purifying the first reactant using ultrasound and a low eutectic solvent to obtain a second reactant, wherein the second reactant is a purified liquid;

[0010] performing extraction and centrifugation on the second reactant to obtain a third reactant;

[0011] The supernatant of the third reactant is collected, and the supernatant is concentrated to obtain the oil-soluble component of the saffron flower without stigma.

[0012] In another aspect of the present invention, preferably,

[0013] The enzyme preparation is used in an amount of 0.05 to 2 wt% of the de-stigmatized saffron flower;

[0014] The enzymatic treatment temperature is 50-60°C;

[0015] The enzymatic treatment time is 60 to 120 minutes;

[0016] The enzymatic treatment method includes spraying the de-stigmatized saffron flower parts with the enzyme preparation.

[0017] In another aspect of the present invention, preferably,

[0018] The enzyme preparation includes lysozyme and pectinase;

[0019] The amount of lysozyme is 1 to 5 wt% of the enzyme preparation;

[0020] The dosage of the pectinase is 0.02-0.1 wt % of the enzyme preparation.

[0021] In another aspect of the present invention, preferably,

[0022] The deep eutectic solvent comprises a hydrogen bond acceptor, a hydrogen bond donor and water;

[0023] The hydrogen bond acceptor includes betaine;

[0024] The hydrogen bond donor includes at least one of urea, xylitol, lactic acid, oxalic acid, glycerol, 1,3-propylene glycol, 1,2-butanediol, 2,3-butanediol, ethylene glycol and n-octanol.

[0025] In another aspect of the present invention, preferably,

[0026] The molar ratio of the hydrogen bond acceptor to the hydrogen bond donor is 1:1 to 1:3;

[0027] The amount of water in the low eutectic solvent is 25-45wt%;

[0028] The liquid-to-solid ratio of the low eutectic solvent to the saffron flower part without stigma is 30-50:1 mL / g.

[0029] In another aspect of the present invention, preferably,

[0030] The power of the ultrasound is 200-600w;

[0031] The extraction time is 20 to 50 minutes;

[0032] The extraction temperature is 50-70°C;

[0033] The purification process includes centrifugation and membrane.

[0034] In another aspect of the present invention, preferably,

[0035] The extraction treatment includes extracting with an extraction solvent;

[0036] The extraction solvent comprises at least one of caprylic / capric triglyceride, isopropyl myristate, isopropyl palmitate and isopropyl isostearate;

[0037] The volume ratio of the extraction solvent to the low eutectic solvent is 1:0.5 to 1:1.5;

[0038] The extraction treatment time is 2 to 4 hours;

[0039] The extraction treatment is performed 2 to 4 times.

[0040] In another aspect of the present invention, preferably,

[0041] The temperature of the centrifugal treatment is 25-30°C;

[0042] The centrifugal speed of the centrifugal treatment is 7000-9000 rpm.

[0043] The centrifugal treatment time is 3 to 8 minutes.

[0044] In another aspect of the present invention, preferably,

[0045] The concentration treatment comprises concentration by a rotary evaporator.

[0046] In another aspect of the present invention, preferably, an oil-soluble component of the flower part of saffron without stigma is used in antioxidant and anti-inflammatory drugs and cosmetics, wherein the oil-soluble component of the flower part of saffron without stigma is obtained by the extraction method as described above.

[0047] (III) Beneficial effects

[0048] The above technical solution of the present invention has the following beneficial technical effects:

[0049] The present invention adopts lysozyme and pectinase as composite enzyme preparation, performs enzymolysis treatment on the saffron flower part with the stigma removed, destroys its cell wall and promotes the release of effective ingredients, releases the oil-soluble components in the cell, and improves the subsequent extraction efficiency. The first reactant after enzymolysis is purified by ultrasound and a low eutectic solvent. Ultrasound can enhance the penetration and dissolution of the solvent to the target component, while the low eutectic solvent can provide a milder extraction environment, reduce the use of traditional organic solvents, and improve the purity of the target component at the same time, and the second reactant after purification is subjected to extraction treatment to further concentrate and extract the target oil-soluble component. Subsequently, centrifugation is performed to thoroughly separate the oil-soluble component from the component insoluble in oil, and a third reactant containing the target component is obtained. Concentration treatment can further remove moisture and impurities, and improve the concentration and purity of the target component. The whole extraction method significantly increases the extraction rate and the content of the active ingredient. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] Figure 1 is an overall flow chart of an embodiment of the present invention;

[0051] Figure 2 It is the response surface diagram and contour diagram of the extraction rate of the present invention;

[0052] Figure 3 4 is a graph showing the free radical scavenging rate of the oil-soluble component of one embodiment of the present invention against DPPH and ABTS. DETAILED DESCRIPTION

[0053] In order to make the purpose, technical scheme and advantages of the present invention clearer, the present invention is further described in detail below in conjunction with specific embodiments and with reference to the accompanying drawings. It should be understood that these descriptions are only exemplary and are not intended to limit the scope of the present invention. In addition, in the following description, the description of well-known structures and technologies is omitted to avoid unnecessary confusion of the concept of the present invention.

[0054] Obviously, the described embodiments are only some embodiments of the present invention, but not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0055] In the description of the present invention, it should be noted that the terms “first”, “second” and “third” are only used for descriptive purposes and cannot be understood as indicating or implying relative importance.

[0056] In addition, the technical features involved in the different embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other.

[0057] Example

[0058] A method for extracting oil-soluble components from saffron flowers without stigma, Figure 1 FIG. 1 shows an overall flow chart of an embodiment of the present invention, as shown in FIG. Figure 1 As shown, including:

[0059] The first reactant is obtained by enzymolysis of the flower part of the saffron stigma removed by using an enzyme preparation; in this embodiment, the amount of the enzyme preparation is 0.05-2wt% of the flower part of the saffron stigma removed; the enzymolysis treatment temperature is 50-60°C; the enzymolysis treatment time is 60-120min; the enzymolysis treatment method includes spraying the flower part of the saffron stigma removed by using the enzyme preparation. The enzyme preparation includes lysozyme and pectinase; the amount of lysozyme is 1-5wt% of the enzyme preparation; the amount of pectinase is 0.02-0.1wt% of the enzyme preparation.

[0060] Lysozyme hydrolyzes the peptidoglycan-like structure in the plant cell wall, destroys the cell wall, increases its permeability, and promotes the mucopolysaccharide in the cell wall of the flower of stigma saffron, thereby releasing more oil-soluble components; at the same time, the cell wall fragments produced after the action may help to adsorb and enrich flavonoids. Pectinase specifically decomposes pectin, which exists in the plant cell wall and intercellular layer and acts as a substance that adheres cells, making the connection between cells loose and the tissue loose, thereby helping to release oil-soluble components. The enzymatic treatment temperature is 50-60℃, which is conducive to the activity of the enzyme preparation. In this temperature range, the enzyme preparation can maintain a high catalytic efficiency, thereby accelerating the decomposition of the flower cell wall and releasing more oil-soluble components. Lysozyme and pectinase make the cell structure loose, opening up channels for betaine and 1,3-propanediol to enter the cell to extract the target components, greatly improving the extraction efficiency. The enzymatic treatment time is 60-120min, which ensures that the enzyme preparation has enough time to fully act on the flower cell wall. By adjusting the enzymatic treatment time, the extraction efficiency of oil-soluble components can be optimized. If the time is too short, the oil-soluble components may not be fully released; if the time is too long, the activity of the enzyme preparation is reduced. The enzymatic treatment method includes spraying the de-stigmatized saffron flower with an enzyme preparation to ensure that the enzyme preparation is evenly distributed on the surface of the flower, thereby improving the uniformity and efficiency of the enzymatic treatment. By spraying, the enzyme preparation can quickly penetrate into the cell wall of the flower and accelerate the decomposition of the cell wall components.

[0061] The first reactant is extracted by ultrasound and a low eutectic solvent to obtain a second reactant, which is a liquid after purification. In this embodiment, the low eutectic solvent includes a hydrogen bond acceptor, a hydrogen bond donor and water. The hydrogen bond acceptor includes betaine. The hydrogen bond donor includes at least one of urea, xylitol, lactic acid, oxalic acid, glycerol, 1,3-propylene glycol, 1,2-butanediol, 2,3-butanediol, ethylene glycol and n-octanol. The molar ratio of the hydrogen bond acceptor to the hydrogen bond donor is 1:1 to 1:3. The amount of water in the low eutectic solvent is 25 to 45 wt%. The liquid-solid ratio of the low eutectic solvent to the stigma-free saffron flower is 30 to 50:1 mL / g. The power of the ultrasound is 200 to 600 w. The extraction time of the extraction treatment is 20 to 50 min. The extraction temperature of the purification treatment is 50 to 70 ° C. The purification treatment includes centrifugation and membrane filtration.

[0062] When ultrasound propagates in liquid, a large number of tiny bubbles are generated and burst quickly. This cavitation effect can produce a strong impact force, which helps to break the cell walls and release more oil-soluble components. The vibration of ultrasound can also produce a strong stirring effect, making the molecules in the liquid move more violently, thereby accelerating the dissolution and diffusion of the solute in the solvent.

[0063] Low eutectic solvents have unique solubility properties and can dissolve a variety of organic and inorganic substances. By selecting suitable hydrogen bond acceptors and hydrogen bond donors, the solubility of the low eutectic solvent can be adjusted so that it can selectively dissolve oil-soluble components to achieve purification. In this embodiment, betaine is used as a hydrogen bond acceptor. Betaine is a zwitterion with good solubility and stability. Urea, xylitol, lactic acid, oxalic acid, glycerol, 1,3-propylene glycol, 1,2-butanediol, 2,3-butanediol, ethylene glycol and n-octanol are used as hydrogen bond donors; betaine and hydrogen bond donors interact through hydrogen bonds to form a stable low eutectic solvent system. The interaction between betaine and hydrogen bond donors reduces the melting point of the solvent, allowing it to remain in a liquid state at a lower temperature, which is not only beneficial to mass transfer and heat transfer during dissolution and extraction, but also has the advantages of low volatility, good solubility, biodegradability and environmental friendliness.

[0064] Betaine is a hydrogen bond acceptor. Because it is a zwitterion, betaine-based low eutectic solvents can significantly improve the extraction rate of oil-soluble components in the flower of de-stigma saffron, and can also maintain the antioxidant activity of the extracted product. Betaine has good biocompatibility and low toxicity, and can be used in cosmetics, foods and drug delivery systems. Betaine has excellent moisturizing properties, can absorb and retain moisture, and enhance the hydration capacity of the skin, making it an ideal moisturizer. In addition, the anti-inflammatory and antioxidant properties of betaine can reduce the discomfort of the skin caused by external stimuli, such as redness, itching and allergies. Betaine can be exerted at low concentrations, has good moisturizing and stabilizing effects, and is non-irritating to the skin and mucous membranes, which makes its application in cosmetics and foods safer, especially suitable for sensitive skin and children's products, thereby reducing the impact of betaine residue problems during the extraction process.

[0065] The amount of water in the low eutectic solvent is 25-45wt%. Water, as a polar solvent, can adjust the polarity and solubility of the low eutectic solvent. By adjusting the amount of water, the purification effect of the low eutectic solvent can be further optimized. The liquid-solid ratio of the low eutectic solvent to the flower of the stigma-free saffron is 30-50:1mL / g, which ensures that the low eutectic solvent can fully contact and dissolve the oil-soluble components in the flower.

[0066] Under the action of ultrasound and deep eutectic solvent, oil-soluble components and some impurity components are extracted from the flower part of saffron without stigma. Extraction time and extraction temperature are key factors affecting extraction efficiency. The extraction time and temperature in this embodiment can accelerate the dissolution and diffusion of target components, thereby improving extraction efficiency.

[0067] The low eutectic solvent composed of betaine and 1,3-propylene glycol combined with the cell structure changes after enzyme treatment can more fully dissolve and extract the target substance and improve the extraction rate. Betaine has the characteristics of protecting the structure and functional stability of biological macromolecules. During the extraction process, it can prevent the plant components from being degraded or denatured due to environmental changes, which helps to maintain the natural structure and activity of the extract and ensure its quality and efficacy. Compared with some traditional extraction methods, the low eutectic solvent composed of betaine and 1,3-propylene glycol combined with the combined treatment method of enzyme treatment is mild and has a certain selectivity, which can more accurately extract the target component, reduce the extraction of unnecessary impurities, and improve the purity of the extract. Betaine and 1,3-propylene glycol are both relatively green and environmentally friendly substances. The low eutectic solvent formed is biodegradable, and has less harm to the environment during use and treatment, which is in line with the development trend of green chemistry. Enzymatic pretreatment makes plant cells easier to extract, which can reduce the use of low eutectic solvents and shorten the extraction time, thereby reducing production costs. After the extraction is completed, the low eutectic solvent of this embodiment can be recovered and reused by appropriate methods, which further improves the economy.

[0068] The purification process includes centrifugation and membrane steps;

[0069] Centrifugation is the process of separating solid residue from the extract after extraction is completed, which helps to remove impurities and undissolved solid components and improve the purity of the extract.

[0070] After centrifugation, the membrane is used to further remove tiny particles and impurities in the extract, ensuring that the final second reactant (the purified liquid) has high purity and transparency.

[0071] Further, in this embodiment, the hydrogen bond donor includes at least one of glycerol, 1,3-propylene glycol and 2,3-butanediol; the molar ratio of hydrogen bond acceptor to hydrogen bond donor is 1:1.9; the amount of water in the low eutectic solvent is 34wt%; when the liquid-solid ratio of the low eutectic solvent to the stigma-free saffron flower is 40:1mL / g, the oil-soluble component extraction rate is high. The second reactant is subjected to extraction treatment and centrifugal treatment to obtain a third reactant; the extraction treatment includes extraction with an extraction solvent; the extraction solvent includes at least one of caprylic / capric triglyceride, isopropyl myristate, isopropyl palmitate and isostearic acid isopropyl ester; the volume ratio of the extraction solvent to the volume of the low eutectic solvent is 1:0.5 to 1:1.5; the extraction treatment time is 2 to 4h; the number of extraction treatments is 2 to 4 times. The temperature of the centrifugal treatment is 25 to 30°C; the centrifugal speed of the centrifugal treatment is 7000 to 9000rpm, and the centrifugal treatment time is 3 to 8min. Extraction is the process of transferring a compound from one solvent to another by utilizing the difference in solubility or partition coefficient of a compound in two immiscible (or slightly soluble) solvents. In this embodiment, the extraction solvent used includes at least one of caprylic / capric triglyceride, isopropyl myristate, isopropyl palmitate and isopropyl isostearate, which have good solubility for oil-soluble components, and the volume ratio of the extraction solvent to the low eutectic solvent is 1:0.5 to 1:1.5, which is to extract most of the oil-soluble components. Centrifugation is the process of separating solid particles or droplets in a mixture by density using centrifugal force. In this embodiment, the temperature of the centrifugation is 25 to 30°C, the centrifugal speed is 7000 to 9000rpm, and the time is 3 to 8 minutes. These parameters are set to achieve effective separation while avoiding damage to heat-sensitive compounds.

[0072] The supernatant of the third reactant is collected, and the supernatant is concentrated to obtain the oil-soluble components of the stigma-free saffron flower. The concentration treatment includes concentration by a rotary evaporator. The concentration treatment is carried out by a rotary evaporator, which rotates the flask at a constant speed through electronic control to increase the evaporation area, and at the same time performs heating diffusion evaporation under negative pressure to accelerate the evaporation rate, so that the reaction product can be separated and purified.

[0073] The invention discloses an application of an oil-soluble component of a flower of saffron without stigma in an antioxidant and anti-inflammatory drug and cosmetic, wherein the oil-soluble component of the flower of saffron without stigma is obtained by the extraction method as described above. The oil-soluble component of the flower of saffron without stigma has significant antioxidant and anti-inflammatory activity because it is rich in flavonoids. Flavonoids can scavenge free radicals and inhibit oxidative stress, thereby playing a role in antioxidant drugs. In anti-inflammatory drugs, these components can reduce inflammatory reactions by inhibiting the release of inflammatory mediators. In cosmetics, the antioxidant properties of the oil-soluble component of the flower of saffron without stigma can help protect the skin from oxidative damage caused by environmental factors and delay skin aging. Its anti-inflammatory properties also help reduce skin inflammation and improve skin conditions such as redness and acne.

[0074] In this embodiment, enzymatic method is first used to pre-treat the flower of saffron without stigma, and the selective release of target components creates favorable conditions for subsequent extraction. Then, betaine-based low eutectic solvent is used to carry out deep extraction, and the oil-soluble components in saffron are fully dissolved and released by virtue of its unique molecular structure and solubility characteristics. Finally, the extract is extracted by grease such as caprylic acid / capric triglyceride, and the target oil-soluble components are accurately enriched. Not only the extraction rate of oil-soluble components of saffron without stigma is greatly improved, but also the biological activity of the extracted product is retained to the greatest extent. At the same time, this method has the outstanding advantages of green environmental protection and low cost, and mild reaction conditions and environmentally friendly materials are used throughout the process, which effectively reduces the impact on the environment and production costs, and helps to promote the further development of saffron related industries.

[0075] Example 1

[0076] The saffron flower part without stigma is enzymatically treated with 0.05wt% enzyme preparation to obtain a first reactant, namely, the petals after enzymatic hydrolysis, in which lysozyme accounts for 1wt% and pectinase accounts for 0.02wt%, the enzymatic hydrolysis temperature is 50°C, and the enzymatic hydrolysis time is 60min.

[0077] A low eutectic solvent is added to the first reactant, the low eutectic solvent is betaine:1,3-propylene glycol (the molar ratio of the two is 1:1), the water content is 25%, the liquid-solid ratio of the low eutectic solvent to the de-stigma saffron flower part is 30:1mL / g, the ultrasonic power is 200w, the extraction temperature is 50°C, the extraction time is 20min, centrifugation and membrane are performed to obtain the second reactant.

[0078] Caprylic / capric triglyceride was added to the second reactant for extraction three times, with a volume ratio of 1:1 to the low eutectic solvent, and each extraction lasted for 3 hours. The extracts were combined and centrifuged at a centrifugal temperature of 25°C, a centrifugal speed of 8000 rpm, and a centrifugal time of 5 minutes. The centrifugal solution was concentrated to obtain the oil-soluble component. The extraction rate of the oil-soluble component was 1.98%.

[0079] Example 2

[0080] The stigma-free saffron flower part was enzymolyzed with 1.03wt% enzyme preparation to obtain a first reactant, namely, petals after enzymolysis, in which lysozyme accounted for 3wt% and pectinase accounted for 0.06wt% in the enzyme preparation, the enzymolysis treatment temperature was 55°C, and the enzymolysis time was 90min.

[0081] A low eutectic solvent is added to the first reactant, the low eutectic solvent is betaine:2,3-butanediol (the molar ratio of the two is 1:2), the water content is 35%, the liquid-solid ratio of the low eutectic solvent to the de-stigma saffron flower part is 40:1mL / g, the ultrasonic power is 400w, the extraction temperature is 60°C, the extraction time is 35min, centrifugation and membrane are performed to obtain the second reactant.

[0082] Isopropyl myristic acid was added to the second reactant for extraction three times, with a volume ratio of 1:1 between isopropyl myristic acid and the low eutectic solvent, and each extraction lasted for 3 hours. The extracts were combined and centrifuged at a centrifugal temperature of 27.5°C, a centrifugal speed of 8000 rpm, and a centrifugal time of 5 minutes. The centrifugal solution was concentrated to obtain the oil-soluble component, wherein the extraction rate of the oil-soluble component was 2.35%.

[0083] Example 3

[0084] The saffron flower part without stigma is enzymatically treated with 0.2wt% enzyme preparation to obtain the first reactant, that is, the petals after enzymatic hydrolysis, the proportion of lysozyme in the enzyme preparation is 5wt%, the proportion of pectinase is 0.1wt%, the enzymatic hydrolysis temperature is 60°C, and the enzymatic hydrolysis time is 120min.

[0085] A low eutectic solvent is added to the first reactant, the low eutectic solvent is betaine:glycerol (the molar ratio of the two is 1:3), the water content is 45%, the liquid-solid ratio of the low eutectic solvent to the de-stigma saffron flower part is 50:1mL / g, the ultrasonic power is 600w, the extraction temperature is 70°C, the extraction time is 50min, centrifugation and membrane are performed to obtain the second reactant.

[0086] Isopropyl isostearate was added to the second reactant for extraction three times, with a volume ratio of isostearate to the low eutectic solvent of 1:1, and each extraction lasted for 3 hours. The extracts were combined and centrifuged at a centrifugal temperature of 30°C, a centrifugal speed of 8000 rpm, and a centrifugal time of 5 minutes. The centrifugal solution was concentrated to obtain the oil-soluble component. The extraction rate of the oil-soluble component was 2.42%.

[0087] Embodiment 4-20

[0088] The stigma-free saffron flower part was enzymolyzed with 1.5wt% enzyme preparation to obtain a first reactant, namely, petals after enzymolysis, in which lysozyme accounted for 3wt% and pectinase accounted for 0.06wt% in the enzyme preparation, the enzymolysis treatment temperature was 50°C, and the enzymolysis time was 120min.

[0089] A low eutectic solvent is added to the first reactant, the low eutectic solvent is betaine, 2,3-butanediol and water, the ultrasonic power is 400w, the extraction temperature is 60°C, the extraction time is 40min, centrifugation and membrane are performed to obtain the second reactant.

[0090] Caprylic acid / capric acid triglyceride was added to the second reactant and extracted three times with a volume ratio of 1:1 to the low eutectic solvent. Each extraction lasted for 3 hours. The extracts were combined and centrifuged at a temperature of 25°C, a speed of 8000 rpm, and a time of 5 minutes. The centrifuged liquid was concentrated to obtain the oil-soluble component.

[0091] Among them, Table 1 shows the experimental parameters and results of Examples 4-20, and the experimental parameters include the molar ratio of 2,3-butanediol: betaine in the deep eutectic solvent, the water content in the deep eutectic solvent, and the liquid-solid ratio of the deep eutectic solvent to the stigma-free saffron flower part. The experimental parameters and results of Examples 4-20 are shown in Table 1:

[0092] Table 1 Experimental parameters and results of Examples 4-20

[0093]

[0094] Design-Expert software was used to analyze the influence of the molar ratio of 2,3-butanediol: betaine in the deep eutectic solvent, the water content in the deep eutectic solvent, and the liquid-solid ratio of the deep eutectic solvent to the stigma-free saffron flower on the extraction rate. The regression model was as follows: Extraction rate (%) = -9.85231 + 1.224505A + 0.31635B + 0.2839C - 0.00825AB + 0.01AC + 0.00065BC - 0.449A 2 -0.00218B 2 -0.004093C 2 .

[0095] The model R 2 The value is 0.99, P<0.01, the model is significant, and the lack of fit term of the experiment is not significant (P=0.78>0.1). The lack of fit term is generally used to verify whether the experimental model is consistent with the theoretical model. The results show that the model has a good fit and the experimental error is not significant. Table 2 shows the regression model and the results of variance analysis. The F value in Table 2 can be used to determine the influence of each single factor on the extraction rate. The order of influence from large to small is A>B>C, that is, the molar ratio of 2,3-butanediol: betaine in the deep eutectic solvent>the water content in the deep eutectic solvent>the liquid-solid ratio of the deep eutectic solvent to the stigma-free saffron flower.

[0096] Table 2 Regression model and variance analysis

[0097]

[0098] Figure 2 The response surface plot and contour plot of the extraction rate are shown, as Figure 2 As shown in Figure 4, the response surface 3D map and contour map can more intuitively reflect the relationship between the independent variable and the response value. The steeper the surface, the more obvious the interaction between the two factors. The closer the shape of the contour line is to an ellipse, the more obvious the interaction between the two factors. As shown in Figure 4, the influence of each single factor on the encapsulation efficiency is as follows: the molar ratio of 2,3-butanediol: betaine in the deep eutectic solvent > the water content in the deep eutectic solvent > the liquid-solid ratio of the deep eutectic solvent to the flower part of the de-stigma saffron. This is consistent with the results of the variance analysis, proving that the regression model is reliable.

[0099] Analysis using Design-Expert8.0.6 software showed that the optimal preparation process was: the molar ratio of betaine: 2,3-butanediol: betaine in the low eutectic solvent was 1.917:1, the water content in the low eutectic solvent was 34.306%, and the liquid-solid ratio of the low eutectic solvent to the saffron flower without stigma was 39.748:1. For the convenience of the experiment, the parameters were modified to: the molar ratio of 2,3-butanediol: betaine was 1:2, the water content in the low eutectic solvent was 34%, and the liquid-solid ratio of the low eutectic solvent to the saffron flower without stigma was 1:40. After verification, it was found that the extraction rate could reach 2.97%, which was consistent with the predicted value as a whole. This shows that the model is reliable and the experimental results are relatively ideal.

[0100] Comparative Example 1

[0101] The only difference from Example 1 is that the enzymolysis is removed, and the other steps remain unchanged.

[0102] Comparative Example 2

[0103] The only difference from Example 1 is that the composite enzyme preparation is replaced by single lysozyme, and the dosage is the same as the total dosage of the enzyme preparation in Example 1.

[0104] Comparative Example 3

[0105] The only difference from Example 1 is that the composite enzyme preparation is replaced by a single cellulase, and the dosage is the same as the total dosage of the enzyme preparation in Example 1.

[0106] Comparative Example 4

[0107] The only difference from Example 1 is that the composite enzyme preparation is replaced by cellulase and lysozyme, and the dosage is the same as the total dosage of the enzyme preparation in Example 1.

[0108] Comparative Example 5

[0109] The only difference from Example 1 is that no low eutectic solvent is used for extraction, only 1,3-propylene glycol is used for extraction, the liquid-solid ratio of petals to 1,3-propylene glycol is 30:1 mL / g, the ultrasonic power is 200w, the extraction temperature is 50°C, and the extraction time is 20min. The other steps remain unchanged.

[0110] According to the method in the document "Zhang Zhihua, et al. Research report on determination of total flavonoid content in corn oil [J]. Grain and Oils, 2002, (09): 44.", the total flavonoid content and extraction rate of the oil-soluble components of the stigma-free saffron flower prepared in Example 1 and the comparative example were determined. Table 3 shows the comparison results of Example 1 and Comparative Examples 1-3. As shown in Table 3,

[0111] Table 3 Comparison results of Example 1 and Comparative Examples 1-3

[0112]

[0113] Compared with Example 1, it can be seen from Comparative Examples 1, 2, 3, and 4 that spraying a certain amount of enzyme preparation helps to extract more oil-soluble components from the flower part of de-stigma saffron and can significantly increase the content of total flavonoids in the extract. When the selected enzymes are lysozyme and pectinase, they can exert a better combined effect with the low eutectic solvent.

[0114] Comparing Comparative Example 5 with Example 1, it can be seen that the low eutectic solvent has a better extraction effect on the oil-soluble components of the flower part of saffron without stigma, and the content of total flavonoids is higher.

[0115] Embodiment 21

[0116] Take a certain amount of DPPH and dissolve it in anhydrous ethanol to prepare a concentration of 2×10 -4 mol / L DPPH ethanol solution, stored at 0-4℃ away from light. Take a certain amount of oil-soluble components and prepare them into a certain concentration with anhydrous ethanol.

[0117] Take 3 mL of the test solution and mix it with 3 mL of DPPH solution, and measure the absorbance at a wavelength of 517 nm (A1). Take 3 mL of anhydrous ethanol and mix it with 3 mL of DPPH solution, and measure the absorbance at a wavelength of 517 nm (A2). Take 3 mL of anhydrous ethanol and mix it with 3 mL of the test solution, and measure the absorbance at a wavelength of 517 nm (A3).

[0118] The DPPH free radical scavenging rate was calculated according to the formula.

[0119]

[0120] Mix 5mL of 7mmol / L ABTS [2,2-azino-di(3-ethyl-benzothiazole-6-sulfonic acid) diammonium salt] and 88μL of 140mmol / L potassium persulfate, and let stand overnight at room temperature in the dark to form a free radical stock solution. Dilute it with anhydrous ethanol to make a working solution before use, so that its absorbance at 734nm is 0.7±0.05. Take 100μL of sample ethanol solution of different concentrations, add 2mL of ABTS solution and shake, and measure the absorbance of the reaction solution at 734nm. 测定 , with A 空白 As a positive control, the absorbance was measured and the ABTS free radical scavenging rate was calculated according to the formula.

[0121]

[0122] Figure 3 The free radical scavenging rate of the oil-soluble component of one embodiment of the present invention on DPPH and ABTS is shown, as shown in FIG. Figure 3As shown in the figure, when the sample concentration is 0.1-0.5 mg / mL, the scavenging ability of DPPH and ABTS increases with the increase of sample concentration. When the sample concentration is 0.5 mg / mL, the scavenging rate of DPPH and ABTS can reach 98.95% and 97.35% respectively.

[0123] Embodiment 22

[0124] This example is based on the LPS (lipopolysaccharide)-induced RAW264.7 macrophage inflammation model to explore the anti-inflammatory activity of the oil-soluble components of the stigma-free saffron flower.

[0125] Cell culture: The cells were cultured in a high-glucose DMEM complete culture medium (containing 100 U / mL penicillin and streptomycin) containing 10% fetal bovine serum in a 5% CO2, 37°C constant temperature incubator. RAW264.7 mouse macrophages in the logarithmic growth phase were used for the experiment.

[0126] The effect of oil-soluble components in the flower of de-stigmatized saffron on cell survival rate was determined: the oil-soluble components were applied to RAW264.7 cells at different concentrations (31.25 μg / mL, 62.5 μg / mL, 125 μg / mL, 250 μg / mL, 500 μg / mL, 1000 μg / mL), and the effect on cell survival rate was detected using the CCK-8 kit.

[0127] Determine the inhibitory effect of the oil-soluble components of destigmatized saffron on the release of NO, IL-8 and TNF-α from RAW264.7 cells induced by LPS: Take the RAW264.7 cell suspension in the logarithmic growth phase and adjust the cell density to 2×10 5 / mL, inoculated in 96-well plates, 100μL in each well, after adherence for 4h, 100μL of each of the two substances with concentrations of 50, 100, and 200μg / mL and 25μL of 1mg / L LPS were added for incubation. A blank group, an LPS group, and an LPS+treatment group were set, with 3 replicates in each group. After culturing for 24h, the NO content in the supernatant was detected by Griess method, and the secretion of IL-8 and TNF-α in the supernatant was detected by ELISA kit.

[0128] Table 4 shows the effect of the concentration of oil-soluble components on the survival rate of RAW264.7 cells. As shown in Table 4, it can be seen from Table 5 that at a concentration of 0-1000 μg / mL, the oil-soluble components of stigma saffron flower are non-toxic to RAW264.7 cells, that is, at a concentration of 0-1000 μg / mL, the substance acts on RAW264.7 cells within a safe concentration range.

[0129] Table 4 Effect of the concentration of oil-soluble components on the survival rate of RAW264.7 cells

[0130]

[0131] Table 5 shows the inhibitory effect of oil-soluble components. As shown in Table 5, the inhibitory effect of oil-soluble components of saffron flower parts on the release of NO, IL-8 and TNF-α induced by LPS in RAW264.7 cells was compared with the blank group. After LPS stimulation, the release of NO, IL-8 and TNF-α in the cell supernatant was significantly increased (P < 0.01). Compared with the LPS group, all concentration groups of the compound can inhibit the release of NO, IL-8 and TNF-α in the cell supernatant, and show concentration dependence (P < 0.05, P < 0.01).

[0132] Table 5 Inhibitory effect of oil-soluble components

[0133]

[0134] The experimental results show that the anti-inflammatory effect of the oil-soluble components of de-stigmatized saffron flowers may be related to the inhibition of the release of NO, IL-8 and TNF-α.

[0135] Embodiment 23

[0136] The invention discloses an essential oil preparation with anti-inflammatory effect, wherein the formula is as follows: 45.25% of caprylic / capric triglyceride, 0.05% of dibutyl hydroxycinnamate hydrogen amyl ester, 0.5% of rape sterols / cetearyl alcohol, 30% of isostearyl isostearate, 0.2% of bisabolol, 0.5% of vitamin E acetate, 20% of decamethylcyclopentasiloxane, 3% of oil-soluble saffron, and 0.5% of macadamia seed oil.

[0137] Preparation method: Weigh 45.25% of caprylic / capric triglyceride, 0.05% of dibutyl hydroxycinnamate, and 0.5% of rape sterols / cetostearyl alcohol in a beaker and heat to 45° C. When they are completely dissolved, cool to room temperature and add the remaining ingredients.

[0138] It should be understood that the above specific embodiments of the present invention are only used to illustrate or explain the principles of the present invention, and do not constitute a limitation of the present invention. Therefore, any modifications, equivalent substitutions, improvements, etc. made without departing from the spirit and scope of the present invention should be included in the protection scope of the present invention. In addition, the appended claims of the present invention are intended to cover all changes and modifications that fall within the scope and boundaries of the appended claims, or the equivalent forms of such scope and boundaries.

[0139] The present invention has been described above with reference to the embodiments of the present invention. However, these embodiments are for illustrative purposes only and are not intended to limit the scope of the present invention. The scope of the present invention is defined by the appended claims and their equivalents. Without departing from the scope of the present invention, a person skilled in the art may make various substitutions and modifications, which should all fall within the scope of the present invention.

[0140] Although the embodiments of the present invention have been described in detail, it should be understood that the various changes, substitutions, and alterations could be made hereto without departing from the spirit and scope of the invention.

[0141] Obviously, the above embodiments are merely examples for the purpose of clear explanation, and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived therefrom are still within the scope of protection of the invention.

Claims

1. A method for extracting oil-soluble components from saffron flowers without stigma, characterized in that: include: Using an enzyme preparation to enzymatically hydrolyze the flower part of saffron without stigma to obtain a first reactant; Extracting and purifying the first reactant using ultrasound and a low eutectic solvent to obtain a second reactant, wherein the second reactant is a purified liquid; performing extraction and centrifugation on the second reactant to obtain a third reactant; The supernatant of the third reactant is collected, and the supernatant is concentrated to obtain the oil-soluble component of the saffron flower without stigma.

2. The extraction method according to claim 1, characterized in that The enzyme preparation is used in an amount of 0.05 to 2 wt% of the de-stigmatized saffron flower; The enzymatic treatment temperature is 50-60°C; The enzymatic treatment time is 60 to 120 minutes; The enzymatic treatment method includes spraying the de-stigmatized saffron flower parts with the enzyme preparation.

3. The extraction method according to claim 1, characterized in that The enzyme preparation includes lysozyme and pectinase; The amount of lysozyme is 1 to 5 wt% of the enzyme preparation; The dosage of the pectinase is 0.02-0.1 wt % of the enzyme preparation.

4. The extraction method according to claim 1, characterized in that The deep eutectic solvent comprises a hydrogen bond acceptor, a hydrogen bond donor and water; The hydrogen bond acceptor is betaine; The hydrogen bond donor includes at least one of urea, xylitol, lactic acid, oxalic acid, glycerol, 1,3-propylene glycol, 1,2-butanediol, 2,3-butanediol, ethylene glycol and n-octanol.

5. The extraction method according to claim 4, characterized in that The molar ratio of the hydrogen bond acceptor to the hydrogen bond donor is 1:1 to 1:3; The amount of water in the low eutectic solvent is 25-45wt%; The liquid-to-solid ratio of the low eutectic solvent to the saffron flower part without stigma is 30-50:1 mL / g.

6. The extraction method according to claim 1, characterized in that The power of the ultrasound is 200-600w; The extraction time is 20 to 50 minutes; The extraction temperature is 50-70°C; The purification process includes centrifugation and membrane.

7. The extraction method according to claim 1, characterized in that The extraction treatment includes extracting with an extraction solvent; The extraction solvent comprises at least one of caprylic / capric triglyceride, isopropyl myristate, isopropyl palmitate and isopropyl isostearate; The volume ratio of the extraction solvent to the low eutectic solvent is 1:0.5 to 1:1.5; The extraction treatment time is 2 to 4 hours; The extraction treatment is performed 2 to 4 times.

8. The extraction method according to claim 1, characterized in that The temperature of the centrifugal treatment is 25-30°C; The centrifugal speed of the centrifugal treatment is 7000-9000 rpm. The centrifugal treatment time is 3 to 8 minutes.

9. The extraction method according to claim 1, characterized in that The concentration treatment comprises concentration by a rotary evaporator.

10. An application of oil-soluble components of saffron flower without stigma in anti-inflammatory and antioxidant drugs and cosmetics, characterized in that: The oil-soluble components of the stigma-free saffron flower are obtained using the extraction method described in any one of claims 1 to 9.

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