A low eutectic solvent microemulsion, a pine mushroom low eutectic microemulsion, and preparation and application thereof

Abstract

The present invention relates to a low eutectic solvent microemulsion, a matsutake low eutectic microemulsion and its preparation and application, and belongs to the field of cosmetic technology. The low eutectic solvent microemulsion of the present invention comprises the following components in weight percentage: 1-10% of a low eutectic solvent, 20-40% of a surfactant, 10-20% of a cosurfactant, and the balance is water. The present invention uses a specific low eutectic solvent and a surfactant, a cosurfactant and water in a specific ratio to prepare a low eutectic solvent microemulsion, and uses the low eutectic solvent microemulsion to extract matsutake, which can simultaneously extract the fat-soluble components and water-soluble components of matsutake, and has high extraction efficiency; after extraction, the active ingredients can also be wrapped inside a microcapsule structure with a uniform particle size to form a matsutake low eutectic microemulsion, thereby improving the stability and bioavailability of the active ingredients and having good antioxidant effects; in addition, the preparation method of the present invention does not use organic solvents, has low energy consumption, is simple and efficient, and is green and environmentally friendly.

Description

Technical Field

[0001] The present invention relates to the technical field of cosmetics, and in particular to a low eutectic solvent microemulsion, a pine twig low eutectic microemulsion, and preparation and application thereof. Background Art

[0002] Matsutake is a rare and precious edible mushroom that is pure and natural, and is known as the "king of mushrooms". Matsutake is delicious and extremely nutritious. Its fruiting bodies contain a variety of bioactive substances, which have antiviral, antibacterial, and anti-inflammatory effects. In addition, it can inhibit tumors, lower blood sugar, and resist radiation. In recent years, as people's attention to natural skin care ingredients continues to increase, Matsutake active ingredients have received widespread attention as a potential cosmetic ingredient.

[0003] Microemulsion is a transparent or translucent uniform dispersion system spontaneously formed by polar phase, non-polar phase and surfactant. Microemulsion overcomes the problems of instability and turbid appearance of conventional emulsions and has ultra-low interfacial tension. Microemulsion is considered to be a microreactor for improving enzyme and chemical reactions. Its huge specific surface area can enhance contact with biological molecules, improve reaction efficiency and shorten reaction time.

[0004] In recent years, low eutectic solvents have replaced the polar phase or non-polar phase of traditional microemulsions to form a new type of low eutectic solvent microemulsion. Low eutectic solvent microemulsions take into account the advantages of both low eutectic solvents and microemulsions. On the one hand, due to the designability of low eutectic solvents, this type of microemulsion has adjustable properties; on the other hand, due to the strong solubility and high thermal stability of low eutectic solvents, the application range of this type of microemulsion is expanded. As a new type of green solvent system, low eutectic solvent microemulsions have broad application prospects in the fields of natural product extraction and separation, drug delivery, biomaterials, etc. Studies have shown that low eutectic solvent microemulsions have the advantages of high efficiency, environmental protection, good mass transfer performance, wide application range and good stability in the extraction of natural products.

[0005] Traditional extraction methods for active ingredients of Matsutake mainly include water extraction, alcohol extraction, ultrasonic extraction, etc. Although these methods are simple and easy to use, the extraction efficiency is relatively low, the active ingredients are easily destroyed, and the organic solvents are easy to pollute the environment. In recent years, some new extraction methods have emerged, such as supercritical carbon dioxide extraction and enzymatic hydrolysis. These methods have high extraction efficiency, but the production cost is also high, and they are not suitable for conventional industrial extraction. Therefore, it is very promising to find simple, efficient and green extraction methods. Summary of the invention

[0006] The purpose of the present invention is to overcome the shortcomings of the prior art and provide a low eutectic solvent microemulsion, a pine twig low eutectic microemulsion and preparation and application thereof.

[0007] To achieve the above purpose, the technical solution adopted by the present invention is:

[0008] In a first aspect, the present invention provides a low eutectic solvent microemulsion, comprising the following components in weight percentage: 1-10% of a low eutectic solvent, 20-40% of a surfactant, 10-20% of a co-surfactant, and the balance being water;

[0009] The low eutectic solvent is prepared from a hydrogen bond donor and a hydrogen bond acceptor, the molar ratio of the hydrogen bond acceptor to the hydrogen bond donor is 1:(1-3), the hydrogen bond acceptor is any one of DL-menthol, lauric acid, choline chloride, betaine, coconut oil and coconut acid, and the hydrogen bond donor is any one of acetic acid, propionic acid, valeric acid, caprylic acid, capric acid, acrylic acid, pyruvic acid, palm kernel oil and babassu seed oil.

[0010] The present invention utilizes specific hydrogen bond donors and hydrogen bond acceptors to prepare a fat-soluble low eutectic solvent, and the fat-soluble low eutectic solvent is used as a non-polar phase to prepare a low eutectic solvent microemulsion with a surfactant, a co-surfactant and water in a specific ratio; the non-fat-soluble low eutectic solvent obtained by replacing it with other hydrogen bond donors and hydrogen bond acceptors cannot be prepared into a low eutectic solvent microemulsion only with a surfactant, a co-surfactant and water. For example, if the low eutectic solvent is replaced with ethylene glycol and L-proline, a water-soluble low eutectic solvent is obtained, which can only be used as a polar phase and cannot be prepared into a low eutectic solvent microemulsion with a surfactant, a co-surfactant and water.

[0011] As a preferred embodiment of the low eutectic solvent microemulsion of the present invention, the molar ratio of the hydrogen bond acceptor to the hydrogen bond donor is 1: 1. Under this ratio, a low eutectic solvent with better solubility can be formed.

[0012] As a preferred embodiment of the deep eutectic solvent microemulsion of the present invention, the hydrogen bond acceptor is any one of DL-menthol and lauric acid, and the hydrogen bond donor is any one of caprylic acid and capric acid. The deep eutectic solvent prepared by the hydrogen bond acceptor and hydrogen bond donor in this range can form a deep eutectic solvent microemulsion with a smaller particle size.

[0013] As a preferred embodiment of the low eutectic solvent microemulsion of the present invention, the hydrogen bond acceptor is lauric acid, and the hydrogen bond donor is capric acid.

[0014] As a preferred embodiment of the low eutectic solvent microemulsion of the present invention, the low eutectic solvent microemulsion comprises the following components in weight percentage: 2-5% of the low eutectic solvent, 20-30% of the surfactant, 10-15% of the co-surfactant, and the balance is water. Within this range, the particle size of the low eutectic solvent microemulsion is smaller.

[0015] As a preferred embodiment of the low eutectic solvent microemulsion of the present invention, the low eutectic solvent microemulsion comprises the following components in weight percentage: 2% low eutectic solvent, 20% surfactant, 10% co-surfactant, and the balance is water.

[0016] As a preferred embodiment of the low eutectic solvent microemulsion of the present invention, the surfactant is one or more of Tween 20, Tween 60, Tween 80, Span 20 (sorbitan monolaurate), Span 60, PEG-20 hydrogenated castor oil, PEG-40 hydrogenated castor oil, and PEG-60 hydrogenated castor oil; the co-surfactant is one or more of ethanol, 1,3-butylene glycol, dipropylene glycol, 1,2-propylene glycol, 1,3-propylene glycol, 1,2-hexanediol, and 1,2-pentanediol.

[0017] As a preferred embodiment of the low eutectic solvent microemulsion of the present invention, the surfactant is one or both of Tween 80 and PEG-40 hydrogenated castor oil; the co-surfactant is one or both of dipropylene glycol and 1,3-propylene glycol.

[0018] As a preferred embodiment of the low eutectic solvent microemulsion of the present invention, the preparation method of the low eutectic solvent is: mixing the hydrogen bond donor and the hydrogen bond acceptor according to a molar ratio, stirring at 70-90° C. until clear and transparent, to obtain the low eutectic solvent.

[0019] As a preferred embodiment of the low eutectic solvent microemulsion of the present invention, the preparation method of the low eutectic solvent is: mixing a hydrogen bond donor and a hydrogen bond acceptor according to a molar ratio, stirring at 80° C. until clear and transparent, to obtain a low eutectic solvent.

[0020] In a second aspect, the present invention provides a method for preparing the above-mentioned low eutectic solvent microemulsion, comprising uniformly mixing the low eutectic solvent, a surfactant and a co-surfactant, adding water and stirring until the system becomes clear and transparent, thereby obtaining the low eutectic solvent microemulsion.

[0021] As a preferred embodiment of the preparation method of the present invention, the uniform mixing is performed in an environment with a temperature of 25-40°C.

[0022] As a preferred embodiment of the preparation method of the present invention, the uniform mixing is carried out in an environment with a temperature of 37°C.

[0023] In a third aspect, the present invention provides a method for preparing a Tricholoma matsutake low eutectic microemulsion, comprising mixing Tricholoma matsutake dry powder with the low eutectic solvent microemulsion, stirring, extracting by reflux at 30-60° C., cooling to room temperature and filtering, and obtaining a filtrate that is the Tricholoma matsutake low eutectic microemulsion.

[0024] The present invention utilizes a low eutectic solvent microemulsion to extract the active ingredients of Tricholoma matsutake. On the one hand, both the fat-soluble active ingredients and the water-soluble active ingredients in Tricholoma matsutake can be extracted. On the other hand, while extracting the active substances, the active substances are microencapsulated to obtain a Tricholoma matsutake low eutectic microemulsion. The preparation method of the present invention greatly improves the extraction rate of the active substances of Tricholoma matsutake, improves the stability and bioavailability of the active substances, and the obtained Tricholoma matsutake low eutectic microemulsion has good antioxidant effect. At the same time, the present invention adopts a low eutectic solvent microemulsion for extraction, does not use an organic solvent, has low energy consumption, and the extraction steps are simple and efficient, and are green and environmentally friendly.

[0025] As a preferred embodiment of the method for preparing the Tricholoma matsutake eutectic microemulsion of the present invention, extraction is performed under reflux at 40°C.

[0026] As a preferred embodiment of the method for preparing the Tricholoma matsutake low eutectic microemulsion of the present invention, the mass volume ratio of the Tricholoma matsutake dry powder to the low eutectic solvent microemulsion is 1 g: (20-40) mL.

[0027] As a preferred embodiment of the method for preparing the Tricholoma matsutake low eutectic microemulsion of the present invention, the mass volume ratio of the Tricholoma matsutake dry powder to the low eutectic solvent microemulsion is 1 g:20 mL.

[0028] As a preferred embodiment of the method for preparing the Tricholoma matsutake eutectic microemulsion of the present invention, the stirring speed is 100-300 r / min, and the extraction time is 2-4 h.

[0029] As a preferred embodiment of the method for preparing the Tricholoma matsutake eutectic microemulsion of the present invention, the stirring speed is 200 r / min and the extraction time is 3 h.

[0030] As a preferred embodiment of the method for preparing the Tricholoma matsutake eutectic microemulsion of the present invention, the method for preparing the Tricholoma matsutake dry powder is as follows: drying and crushing Tricholoma matsutake fruiting bodies, and passing through a 60-100 mesh sieve to obtain Tricholoma matsutake dry powder. Preferably, passing through an 80 mesh sieve.

[0031] In a fourth aspect, the present invention provides a Tricholoma matsutake eutectic microemulsion prepared by the method for preparing the Tricholoma matsutake eutectic microemulsion.

[0032] In a fifth aspect, the present invention provides the use of the Tricholoma matsutake eutectic microemulsion in the preparation of skin care products. The Tricholoma matsutake eutectic microemulsion prepared by the present invention has good antioxidant properties and can be prepared into skin care products.

[0033] Compared with the prior art, the present invention has the following beneficial effects:

[0034] The present invention uses a specific low eutectic solvent and a surfactant, a co-surfactant and water in a specific ratio to prepare a low eutectic solvent microemulsion, and uses the low eutectic solvent microemulsion to extract matsutake, which can extract both the fat-soluble components and the water-soluble components of matsutake, and has high extraction efficiency; after extraction, the active ingredients can be wrapped inside a microcapsule structure with a uniform particle size to form a matsutake low eutectic microemulsion, which can improve the stability of the active ingredients on the one hand, and improve the bioavailability of the active ingredients on the other hand, and has good antioxidant effect; in addition, the preparation method of the present invention does not use an organic solvent, has low energy consumption, has the advantages of simplicity, high efficiency and green environmental protection, provides a basis for preparing a high-quality matsutake low eutectic microemulsion, and provides a way of thinking for the application of matsutake low eutectic microemulsion in natural skin care products. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 This is the appearance of the deep eutectic solvent microemulsion prepared in Example 1 of the present invention;

[0036] Figure 2 The particle size distribution diagram of the deep eutectic solvent microemulsion and the Tricholoma matsutake deep eutectic microemulsion of Example 1 of the present invention;

[0037] Figure 3 The appearance of the Tricholoma matsutake eutectic microemulsion of Example 1 and Comparative Example 8, the Tricholoma matsutake water extract of Comparative Example 9, the Tricholoma matsutake alcohol extract of Comparative Example 10, the Tricholoma matsutake microemulsion of Comparative Example 11, and the Tricholoma matsutake fat emulsion of Comparative Example 13 of the present invention;

[0038] Figure 4 This is an electron microscope image of the Tricholoma matsutake eutectic microemulsion of Example 1 of the present invention;

[0039] Figure 5 The total polysaccharide, total polyphenol and total flavonoid retention rates of the pine twig eutectic microemulsion of Example 1 of the present invention, the pine twig water extract of Comparative Example 9 and the pine twig alcohol extract of Comparative Example 10 after being illuminated for different days, A represents the total polysaccharide retention rate, B represents the total polyphenol retention rate, and C represents the total flavonoid retention rate;

[0040] Figure 6 The total polysaccharide intradermal retention results of the Tricholoma matsutake eutectic microemulsion of Example 1 of the present invention and the Tricholoma matsutake water extract of Comparative Example 9 at different times;

[0041] Figure 7 The DPPH radical scavenging results of the Tricholoma matsutake eutectic microemulsion of Example 1 of the present invention, the Tricholoma matsutake water extract of Comparative Example 9, and the Tricholoma matsutake alcohol extract of Comparative Example 10;

[0042] Figure 8 The hydroxyl radical scavenging results of the pine twig eutectic microemulsion of Example 1 of the present invention, the pine twig water extract of Comparative Example 9, and the pine twig alcohol extract of Comparative Example 10;

[0043] Fig. 9 The fluorescence images of the control group in Test Example 8 of the present invention and Example 1, Comparative Example 9 and Comparative Example 10 in the experimental group;

[0044] Fig.10 It is a bar graph of the relative IOD values ​​of the control group in Test Example 8 of the present invention and Example 1, Comparative Example 9, and Comparative Example 10 in the experimental group. DETAILED DESCRIPTION

[0045] In order to better illustrate the purpose, technical solutions and advantages of the present invention, the present invention will be further described below in conjunction with specific embodiments.

[0046] Unless otherwise specified, other materials, reagents, etc. used in the examples can be obtained from commercial sources.

[0047] Example 1

[0048] 1. The preparation of a low eutectic solvent microemulsion comprises the following steps:

[0049] a. Preparation of a low eutectic solvent: lauric acid as a hydrogen bond acceptor and capric acid as a hydrogen bond donor are mixed in a round-bottom flask at a molar ratio of 1:1 and stirred at 80° C. under a constant temperature magnetic stirrer until the mixture becomes a clear and transparent liquid to obtain a lauric acid / capric acid low eutectic solvent;

[0050] b. Preparation of low eutectic solvent microemulsion: 2% lauric acid / capric acid low eutectic solvent, 20% Tween 80 and 10% dipropylene glycol were added to a container, placed in a 37°C constant temperature water bath and stirred to mix, and 68% water was added under slow stirring until the system became clear and transparent to obtain a low eutectic solvent microemulsion. The appearance of the low eutectic solvent microemulsion prepared in this example is as follows: Figure 1 shown.

[0051] 2. The preparation of Tricholoma matsutake eutectic microemulsion comprises the following steps:

[0052] A. Pretreatment of Tricholoma matsutake: dry and slice the Tricholoma matsutake fruiting bodies, grind them through an 80-mesh sieve with a grinder to obtain Tricholoma matsutake dry powder;

[0053] B. Preparation of Tricholoma matsutake eutectic microemulsion: 20 g of Tricholoma matsutake dry powder was placed in a round-bottom flask, and 400 mL of the low eutectic solvent microemulsion prepared in this example was added. The mixture was heated under reflux in a 40°C water bath with stirring at 200 r / min for 3 h, cooled to room temperature, and filtered to obtain Tricholoma matsutake eutectic microemulsion.

[0054] Example 2

[0055] 1. The preparation of a low eutectic solvent microemulsion comprises the following steps:

[0056] a. Preparation of a low eutectic solvent: DL-menthol as a hydrogen bond acceptor and octanoic acid as a hydrogen bond donor are mixed in a round-bottom flask at a molar ratio of 1:1 and stirred under a constant temperature magnetic stirrer at 80° C. until the mixture becomes a clear and transparent liquid to obtain a DL-menthol / octanoic acid low eutectic solvent;

[0057] b. Preparation of low eutectic solvent microemulsion: Add 4% DL-menthol / octanoic acid low eutectic solvent, 30% PEG-40 hydrogenated castor oil and 15% 1,3-propylene glycol into a container, place in a constant temperature water bath at 37°C and stir to mix, add 51% water while slowly stirring until the system becomes clear and transparent to obtain a low eutectic solvent microemulsion.

[0058] 2. The preparation of Tricholoma matsutake eutectic microemulsion comprises the following steps:

[0059] A. Pretreatment of Tricholoma matsutake: dry and slice the Tricholoma matsutake fruiting bodies, grind them through an 80-mesh sieve with a grinder to obtain Tricholoma matsutake dry powder;

[0060] B. Preparation of Tricholoma matsutake eutectic microemulsion: 20 g of Tricholoma matsutake dry powder was placed in a round-bottom flask, and 400 mL of the low eutectic solvent microemulsion prepared in this example was added. The mixture was heated under reflux in a 40°C water bath with stirring at 200 r / min for 3 h, cooled to room temperature, and filtered to obtain Tricholoma matsutake eutectic microemulsion.

[0061] Example 3

[0062] 1. The preparation of a low eutectic solvent microemulsion comprises the following steps:

[0063] a. Preparation of a low eutectic solvent: betaine as a hydrogen bond acceptor and propionic acid as a hydrogen bond donor are mixed in a round-bottom flask at a molar ratio of 1:1 and stirred at 80° C. under a constant temperature magnetic stirrer until the mixture becomes a clear and transparent liquid to obtain a betaine / propionic acid low eutectic solvent;

[0064] b. Preparation of low eutectic solvent microemulsion: Add 5% betaine / propionic acid low eutectic solvent, 30% Tween 20 and 15% ethanol into a container, place in a constant temperature water bath at 37°C and stir to mix, add 50% water while slowly stirring until the system becomes clear and transparent to obtain a low eutectic solvent microemulsion.

[0065] 2. The preparation of Tricholoma matsutake eutectic microemulsion comprises the following steps:

[0066] A. Pretreatment of Tricholoma matsutake: dry and slice the Tricholoma matsutake fruiting bodies, grind them through a 60-mesh sieve with a grinder to obtain Tricholoma matsutake dry powder;

[0067] B. Preparation of Tricholoma matsutake eutectic microemulsion: 20 g of Tricholoma matsutake dry powder was placed in a round-bottom flask, and 400 mL of the low eutectic solvent microemulsion prepared in this example was added. The mixture was heated under reflux in a 50°C water bath under stirring at 200 r / min for 4 h, cooled to room temperature, and filtered to obtain Tricholoma matsutake eutectic microemulsion.

[0068] Example 4

[0069] 1. The preparation of a low eutectic solvent microemulsion comprises the following steps:

[0070] a. Preparation of a low eutectic solvent: choline chloride as a hydrogen bond acceptor and acetic acid as a hydrogen bond donor are mixed in a round-bottom flask at a molar ratio of 1:1 and stirred at 80° C. under a constant temperature magnetic stirrer until the mixture becomes a clear and transparent liquid to obtain a choline chloride / acetic acid low eutectic solvent;

[0071] b. Preparation of low eutectic solvent microemulsion: Add 10% choline chloride / acetic acid low eutectic solvent, 40% Tween 60 and 20% 1,3-butanediol into a container, place in a constant temperature water bath at 37°C and stir to mix, add 30% water while slowly stirring until the system becomes clear and transparent to obtain a low eutectic solvent microemulsion.

[0072] 2. The preparation of Tricholoma matsutake eutectic microemulsion comprises the following steps:

[0073] A. Pretreatment of Tricholoma matsutake: dry and slice the Tricholoma matsutake fruiting bodies, and grind them through a 100-mesh sieve with a grinder to obtain Tricholoma matsutake dry powder;

[0074] B. Preparation of Tricholoma matsutake eutectic microemulsion: 20 g of Tricholoma matsutake dry powder was placed in a round-bottom flask, and 400 mL of the low eutectic solvent microemulsion prepared in this example was added. The mixture was heated under reflux in a water bath at 60°C for 2 h under stirring at 200 r / min, cooled to room temperature, and filtered to obtain Tricholoma matsutake eutectic microemulsion.

[0075] Example 5

[0076] 1. The preparation of a low eutectic solvent microemulsion comprises the following steps:

[0077] a. Preparation of a low eutectic solvent: lauric acid as a hydrogen bond acceptor and valeric acid as a hydrogen bond donor are mixed in a round-bottom flask at a molar ratio of 1:1 and stirred at 70° C. under a constant temperature magnetic stirrer until the mixture becomes a clear and transparent liquid to obtain a lauric acid / valeric acid low eutectic solvent;

[0078] b. Preparation of low eutectic solvent microemulsion: Add 6% lauric acid / valeric acid low eutectic solvent, 35% Span 20 and 20% 1,2-hexanediol into a container, place in a constant temperature water bath at 37°C and stir to mix, add 39% water while slowly stirring until the system becomes clear and transparent to obtain a low eutectic solvent microemulsion.

[0079] 2. The preparation of Tricholoma matsutake eutectic microemulsion comprises the following steps:

[0080] A. Pretreatment of Tricholoma matsutake: dry and slice the Tricholoma matsutake fruiting bodies, grind them through an 80-mesh sieve with a grinder to obtain Tricholoma matsutake dry powder;

[0081] B. Preparation of Tricholoma matsutake eutectic microemulsion: 20 g of Tricholoma matsutake dry powder was placed in a round-bottom flask, and 400 mL of the low eutectic solvent microemulsion prepared in this example was added. The mixture was heated under reflux in a 30°C water bath with stirring at 300 r / min for 4 h, cooled to room temperature, and filtered to obtain Tricholoma matsutake eutectic microemulsion.

[0082] Example 6

[0083] 1. The preparation of a low eutectic solvent microemulsion comprises the following steps:

[0084] a. Preparation of a low eutectic solvent: DL-menthol as a hydrogen bond acceptor and acrylic acid as a hydrogen bond donor are mixed in a round-bottom flask at a molar ratio of 1:1 and stirred at 80° C. under a constant temperature magnetic stirrer until the mixture becomes a clear and transparent liquid to obtain a DL-menthol / acrylic acid low eutectic solvent;

[0085] b. Preparation of low eutectic solvent microemulsion: Add 8% DL-menthol / acrylic acid low eutectic solvent, 40% PEG-20 hydrogenated castor oil and 10% 1,2-pentanediol into a container, place in a constant temperature water bath at 37°C and stir to mix, add 42% water while slowly stirring until the system becomes clear and transparent to obtain a low eutectic solvent microemulsion.

[0086] 2. The preparation of Tricholoma matsutake eutectic microemulsion comprises the following steps:

[0087] A. Pretreatment of Tricholoma matsutake: dry and slice the Tricholoma matsutake fruiting bodies, grind them through an 80-mesh sieve with a grinder to obtain Tricholoma matsutake dry powder;

[0088] B. Preparation of Tricholoma matsutake eutectic microemulsion: 20 g of Tricholoma matsutake dry powder was placed in a round-bottom flask, and 600 mL of the low eutectic solvent microemulsion prepared in this example was added. The mixture was heated under reflux in a 40°C water bath with stirring at 100 r / min for 3 h, cooled to room temperature, and filtered to obtain Tricholoma matsutake eutectic microemulsion.

[0089] Example 7

[0090] 1. The preparation of a low eutectic solvent microemulsion comprises the following steps:

[0091] a. Preparation of a low eutectic solvent: lauric acid as a hydrogen bond acceptor and pyruvic acid as a hydrogen bond donor are mixed in a round-bottom flask at a molar ratio of 1:1 and stirred at 90° C. under a constant temperature magnetic stirrer until the mixture becomes a clear and transparent liquid to obtain a lauric acid / pyruvic acid low eutectic solvent;

[0092] b. Preparation of low eutectic solvent microemulsion: Add 1% lauric acid / pyruvic acid low eutectic solvent, 20% PEG-60 hydrogenated castor oil and 10% dipropylene glycol into a container, place in a constant temperature water bath at 37°C and stir to mix, add 69% water while slowly stirring until the system becomes clear and transparent to obtain a low eutectic solvent microemulsion.

[0093] 2. The preparation of Tricholoma matsutake eutectic microemulsion comprises the following steps:

[0094] A. Pretreatment of Tricholoma matsutake: dry and slice the Tricholoma matsutake fruiting bodies, grind them through an 80-mesh sieve with a grinder to obtain Tricholoma matsutake dry powder;

[0095] B. Preparation of Tricholoma matsutake eutectic microemulsion: 20 g of Tricholoma matsutake dry powder was placed in a round-bottom flask, and 800 mL of the low eutectic solvent microemulsion prepared in this example was added. The mixture was heated under reflux in a 40°C water bath with stirring at 200 r / min for 3 h, cooled to room temperature, and filtered to obtain Tricholoma matsutake eutectic microemulsion.

[0096] Example 8

[0097] 1. The preparation of the low eutectic solvent microemulsion is the same as that in Example 1, except that in step a, the molar ratio of lauric acid to capric acid is replaced from 1:1 to 1:2.

[0098] 2. The preparation of the Tricholoma matsutake eutectic microemulsion is the same as that in Example 1, except that in step B, the eutectic solvent microemulsion is replaced by the eutectic solvent microemulsion prepared in this example.

[0099] Example 9

[0100] 1. The preparation of the low eutectic solvent microemulsion is the same as that in Example 1, except that in step a, the molar ratio of lauric acid to capric acid is replaced from 1:1 to 1:3.

[0101] 2. The preparation of the Tricholoma matsutake eutectic microemulsion is the same as that in Example 1, except that in step B, the eutectic solvent microemulsion is replaced by the eutectic solvent microemulsion prepared in this example.

[0102] Example 10

[0103] 1. The preparation of the low eutectic solvent microemulsion is the same as that in Example 1, except that in step a, lauric acid is replaced by coconut oil, and capric acid is replaced by palm kernel oil.

[0104] 2. The preparation of the Tricholoma matsutake eutectic microemulsion is the same as that in Example 1, except that in step B, the eutectic solvent microemulsion is replaced by the eutectic solvent microemulsion prepared in this example.

[0105] Embodiment 11

[0106] 1. The preparation of the low eutectic solvent microemulsion is the same as that in Example 1, except that in step a, lauric acid is replaced by coconut acid, and capric acid is replaced by babassu seed oil.

[0107] 2. The preparation of the Tricholoma matsutake eutectic microemulsion is the same as that in Example 1, except that in step B, the eutectic solvent microemulsion is replaced by the eutectic solvent microemulsion prepared in this example.

[0108] Comparative Example 1

[0109] 1. The preparation of the low eutectic solvent microemulsion is the same as that of Example 1, except that in step a, the hydrogen bond acceptor lauric acid is replaced by L-carnitine.

[0110] The results showed that L-carnitine remained in a crystalline state and was immiscible with lauric acid, making it impossible to prepare a low eutectic solvent, form a low eutectic solvent microemulsion, and subsequently extract the active ingredients of Tricholoma matsutake.

[0111] Comparative Example 2

[0112] 1. The preparation of the low eutectic solvent microemulsion is the same as that in Example 1, except that in step a, the hydrogen bond donor decanoic acid is replaced by L-proline.

[0113] The results showed that L-proline remained in a crystalline state and was immiscible with lauric acid, making it impossible to prepare a low eutectic solvent, form a low eutectic solvent microemulsion, and subsequently extract the active ingredients of Tricholoma matsutake.

[0114] Comparative Example 3

[0115] 1. The preparation of the low eutectic solvent microemulsion is the same as that in Example 1, except that in step a, the molar ratio of lauric acid to capric acid is replaced from 1:1 to 1:0.5.

[0116] The results showed that lauric acid crystals precipitated during the cooling process to room temperature, and it was impossible to prepare a low eutectic solvent, form a low eutectic solvent microemulsion, and subsequently extract the active ingredients of Tricholoma matsutake.

[0117] Comparative Example 4

[0118] 1. The preparation of the low eutectic solvent microemulsion is the same as that in Example 1, except that in step a, the molar ratio of lauric acid to capric acid is replaced from 1:1 to 1:4.

[0119] The results showed that lauric acid crystals precipitated during the cooling process to room temperature, and it was impossible to prepare a low eutectic solvent, form a low eutectic solvent microemulsion, and subsequently extract the active ingredients of Tricholoma matsutake.

[0120] Comparative Example 5

[0121] 1. The preparation of the low eutectic solvent microemulsion is the same as that in Example 1, except that in step b, the content of dipropylene glycol is replaced from 10% to 20%.

[0122] The results showed that the prepared liquid was obviously stratified, and it was impossible to prepare a low eutectic solvent microemulsion, and it was impossible to perform subsequent extraction of the active ingredients of Tricholoma matsutake.

[0123] Comparative Example 6

[0124] 1. The preparation of the low eutectic solvent microemulsion is the same as that in Example 1, except that in step b, the content of the lauric acid / capric acid low eutectic solvent is replaced from 2% to 15%.

[0125] The results showed that the lauric acid / capric acid low eutectic solvent floated on the liquid surface, making it impossible to prepare a low eutectic solvent microemulsion and subsequently extract the active ingredients of Tricholoma matsutake.

[0126] Comparative Example 7

[0127] 1. The preparation of the low eutectic solvent microemulsion is the same as that in Example 1, except that in step a, the hydrogen bond acceptor lauric acid is replaced by ethylene glycol, and the hydrogen bond donor capric acid is replaced by L-proline.

[0128] The results showed that the ethylene glycol / L-proline low eutectic solvent was a water-soluble liquid. It was used as a polar phase to prepare a mixed solution with Tween 80, dipropylene glycol and water. However, a clear and transparent low eutectic solvent microemulsion could not be prepared, and subsequent extraction of the active ingredients of Tricholoma matsutake could not be performed.

[0129] Comparative Example 8

[0130] 1. Preparation of low eutectic solvent microemulsion is the same as in Example 1.

[0131] 2. The preparation of the Tricholoma matsutake eutectic microemulsion is the same as that in Example 1, except that in step B, the water bath heating temperature is replaced from 40°C to 70°C.

[0132] Comparative Example 9

[0133] A method for preparing a Tricholoma matsutake water extract comprises the following steps:

[0134] A. Pretreatment of Tricholoma matsutake: same as in Example 1;

[0135] B. Preparation of Tricholoma matsutake aqueous extract: The same as Example 1, except that in step B, the low eutectic solvent microemulsion is replaced with water to obtain Tricholoma matsutake aqueous extract.

[0136] Comparative Example 10

[0137] A method for preparing a Tricholoma matsutake alcohol extract comprises the following steps:

[0138] A. Pretreatment of Tricholoma matsutake: same as in Example 1;

[0139] B. Preparation of Tricholoma matsutake alcohol extract: The same as Example 1, except that in step B, the low eutectic solvent microemulsion is replaced with 60% ethanol to obtain Tricholoma matsutake alcohol extract.

[0140] Comparative Example 11

[0141] The traditional microemulsion was used to extract the matsutake mushroom and prepare the matsutake mushroom microemulsion.

[0142] 1. Preparation of traditional microemulsion: Same as Example 1, except that in step b, the lauric acid / capric acid low eutectic solvent is replaced by lauric acid.

[0143] 2. Preparation of Tricholoma matsutake microemulsion: The same as Example 1, except that in step B, the low eutectic solvent microemulsion is replaced with the traditional microemulsion prepared in this comparative example to obtain Tricholoma matsutake microemulsion.

[0144] Comparative Example 12

[0145] The traditional microemulsion was used to extract the matsutake mushroom and prepare the matsutake mushroom microemulsion.

[0146] 1. Preparation of traditional microemulsion: Same as Example 1, except that in step b, the lauric acid / capric acid low eutectic solvent is replaced by capric acid.

[0147] 2. The preparation of Tricholoma matsutake microemulsion comprises the following steps:

[0148] A. Pretreatment of Tricholoma matsutake: same as in Example 1;

[0149] B. Preparation of Tricholoma matsutake microemulsion: The same as Example 1, except that in step B, the low eutectic solvent microemulsion is replaced with the traditional microemulsion prepared in this comparative example to obtain Tricholoma matsutake microemulsion.

[0150] Comparative Example 13

[0151] The matsutake fat emulsion was prepared by extracting matsutake fat emulsion.

[0152] 1. The preparation of fat emulsion comprises the following steps:

[0153] 1.2% egg yolk lecithin and 0.02% tocopherol were added to 6% caprylic / capric triglyceride and stirred at 60°C until completely dissolved as the oil phase; 0.03% sodium oleate and 2.5% glycerol were added to 90.25% deionized water and stirred at 60°C until completely dissolved as the water phase; the oil phase was slowly added to the water phase under constant temperature and high-speed shear conditions at 60°C, the shear speed was 12000 rpm / min, and the shearing was performed for 10 min to obtain colostrum, which was then subjected to high-pressure homogenization at a homogenization pressure of 700 bar, and the homogenization was repeated 3 times to obtain a stable fat emulsion.

[0154] 2. Preparation of Tricholoma matsutake fat emulsion: The same as Example 1, except that in step B, the low eutectic solvent microemulsion is replaced with the fat emulsion prepared in this comparative example to obtain Tricholoma matsutake fat emulsion.

[0155] Test Example 1

[0156] 1. Methods

[0157] The particle size and PDI of the low eutectic solvent microemulsion (liquid before extracting pine mushroom) and the pine mushroom low eutectic microemulsion (liquid after extracting pine mushroom) of Examples 1-11 of the present invention and Comparative Example 8 were respectively detected by a laser particle size analyzer, and the particle size and PDI of the traditional microemulsion, fat emulsion (liquid before extracting pine mushroom) and pine mushroom water extract, pine mushroom alcohol extract, pine mushroom microemulsion, and pine mushroom fat emulsion (liquid after extracting pine mushroom) of Comparative Examples 9-13 were respectively detected. The electron microscope structure of the liquid after extracting pine mushroom was detected by transmission electron microscopy. Comparative Examples 1-4 could not prepare a low eutectic solvent, and the subsequent preparation of a low eutectic solvent microemulsion could not be carried out, so the particle size detection could not be carried out. Comparative Examples 5-7 could not prepare a low eutectic solvent microemulsion, so the particle size detection could not be carried out.

[0158] 2. Results

[0159] (1) The particle size and PDI test results are shown in Table 1 below, where the particle size distribution diagrams of the low eutectic solvent microemulsion of Example 1 and the Tricholoma matsutake low eutectic microemulsion are shown in Table 1. Figure 2 As shown,

[0160] Table 1

[0161]

[0162] Note: / in the table indicates that particle size detection is not possible.

[0163] The results in Table 1 show that before extracting Tricholoma matsutake, the low eutectic solvent microemulsions of Examples 1-11 and Comparative Example 8 and the traditional microemulsions of Comparative Examples 11 and 12 have a particle size of 10-20 nm, a particle size PDI of less than 0.25, a small particle size and a uniform particle size distribution; after extracting Tricholoma matsutake, the active ingredients of Tricholoma matsutake enter the microcapsule structure, and the particle size of the formed Tricholoma matsutake low eutectic microemulsion / Tricholoma matsutake microemulsion becomes larger, with a particle size of 80-100 nm, a particle size PDI of less than 0.25, a small particle size and a uniform particle size distribution. Comparative Examples 9 and 10 form water extracts and alcohol extracts of Tricholoma matsutake, and cannot form a microcapsule structure, so the particle size cannot be detected. Before extracting Matsutake, the particle size of the fat emulsion of Comparative Example 13 was about 182 nm, the PDI of the particle size was 0.19, and the particle size distribution was uniform. After extracting Matsutake, the particle size of the obtained Matsutake fat emulsion increased significantly (reaching about 2400 nm) and the PDI of the particle size also increased significantly (reaching 0.36), indicating that the uniformity of the extract obtained by extracting the active ingredients of Matsutake using fat emulsion became poor.

[0164] (2) The appearance of the eutectic microemulsion of Tricholoma matsutake of Example 1 and Comparative Example 8, the aqueous extract of Tricholoma matsutake of Comparative Example 9, the alcohol extract of Tricholoma matsutake of Comparative Example 10, the microemulsion of Tricholoma matsutake of Comparative Example 11, and the fat emulsion of Tricholoma matsutake of Comparative Example 13 is as follows: Figure 3 As shown, the results show that the pine twig eutectic microemulsion of Example 1 is a clear and transparent brown liquid; the pine twig eutectic microemulsion of Comparative Example 8 is a clear and transparent dark brown liquid, indicating that the extraction temperature is too high, resulting in serious discoloration of the active substances in the pine twigs, and the appearance of the extracted pine twig eutectic microemulsion is unstable; the pine twig water extract of Comparative Example 9 is a translucent brown liquid; the pine twig alcohol extract of Comparative Example 10 and the pine twig microemulsion of Comparative Example 11 are clear and transparent light brown liquids; the pine twig fat emulsion of Comparative Example 13 is an opaque liquid, and stratification occurs, indicating that the stable system of the fat emulsion is destroyed, indicating that the fat emulsion is not suitable for extracting active substances in pine twigs.

[0165] (3) The electron microscopic image of the Tricholoma matsutake eutectic microemulsion of Example 1 is as follows: Figure 4 As shown, the results showed that the low eutectic microemulsion of Tricholoma matsutake was a uniform microcapsule structure, indicating that after the Tricholoma matsutake was extracted by the low eutectic solvent microemulsion, the active ingredients of Tricholoma matsutake entered the microcapsule structure and formed a uniform system.

[0166] Test Example 2

[0167] The extraction rates of water-soluble active ingredients (total polysaccharides) and fat-soluble active ingredients (total polyphenols, total flavonoids) in the low eutectic microemulsions of Tricholoma matsutake of Examples 1-11 of the present invention and Comparative Example 8 (low eutectic solvent microemulsion extraction), the water extract of Tricholoma matsutake of Comparative Example 9 (water extraction), the alcohol extract of Tricholoma matsutake of Comparative Example 10 (alcohol extraction), the microemulsions of Tricholoma matsutake of Comparative Examples 11 and 12 (traditional microemulsion extraction), and the fat emulsion of Tricholoma matsutake of Comparative Example 13 (fat emulsion extraction) were tested.

[0168] The total polysaccharide content was detected by the phenol-sulfuric acid method, and the total polysaccharide extraction rate was calculated according to the following formula:

[0169] Total polysaccharide extraction rate (%) = total polysaccharide content × volume of deep eutectic solvent microemulsion / mass of pine mushroom dry powder × 100%;

[0170] The total polyphenol content was detected by the Folin-Ciocalteu method, and the total polyphenol extraction rate was calculated according to the following formula:

[0171] Total polyphenol extraction rate (mg / g) = total polyphenol content × volume of deep eutectic solvent microemulsion / mass of pine mushroom dry powder;

[0172] The total flavonoid content was detected by sodium nitrite-aluminum nitrate colorimetry, and the total flavonoid extraction rate was calculated according to the following formula:

[0173] Total flavonoid extraction rate (mg / g) = total flavonoid content × volume of low eutectic solvent microemulsion / mass of Tricholoma matsutake dry powder.

[0174] The results of total polysaccharide, total polyphenol and total flavonoid extraction rates are shown in Table 2.

[0175] Table 2

[0176]

[0177] The results in Table 2 show that the methods of extracting using the low eutectic solvent microemulsion of the present invention (Examples 1-11, Comparative Example 8), the traditional microemulsion (Comparative Examples 11, 12) and the traditional solvent (Comparative Examples 9, 10) can all extract the water-soluble and fat-soluble active ingredients in Tricholoma matsutake; compared with the three extraction methods, the traditional solvent has the lowest efficiency in extracting total polysaccharides from Tricholoma matsutake, the traditional microemulsion can improve the extraction efficiency of total polysaccharides, but the low eutectic solvent microemulsion has the highest extraction efficiency for total polysaccharides, indicating that the low eutectic solvent microemulsion can further improve the extraction efficiency of total polysaccharides on the basis of the traditional microemulsion, but when the extraction temperature reaches 70°C, the extraction rate of total polysaccharides begins to decrease, indicating that the low eutectic solvent microemulsion of the present invention has the best effect on extracting the active ingredients of Tricholoma matsutake in the range of 30-60°C. When using fat emulsion (Comparative Example 13) to extract active substances from Tricholoma matsutake, only total polysaccharides and total polyphenols in Tricholoma matsutake can be extracted, and the extraction rate is very low, and total flavonoids cannot be extracted, which once again proves that fat emulsion is not suitable for extracting active substances from Tricholoma matsutake.

[0178] Test Example 3

[0179] The stability of the Tricholoma matsutake eutectic microemulsion (eutectic solvent microemulsion extraction) of Examples 1-11, the Tricholoma matsutake water extract (water extraction) of Comparative Example 9, and the Tricholoma matsutake alcohol extract (alcohol extraction) of Comparative Example 10 was tested. Each solution was placed under light conditions (light intensity 4500±500lux), and the total polysaccharide, total polyphenol and total flavonoid contents on day 0, day 30, day 60, and day 90 were detected respectively (the determination method of the total polysaccharide, total polyphenol and total flavonoid contents was the same as that of Test Example 2), and the retention rate was calculated according to the following formula:

[0180]

[0181] The test results showed that after 90 days of observation under light conditions, the retention rate of total polysaccharides of the Tricholoma matsutake eutectic microemulsion of Examples 1-11 was 96.51% to 97.20%, the retention rate of total polyphenols was 72.4% to 78%, and the retention rate of total flavonoids was 79.86% to 82.09%.

[0182] The retention rates of total polysaccharides, total polyphenols and total flavonoids of the pine twig eutectic microemulsion of Example 1, the pine twig water extract of Comparative Example 9 and the pine twig alcohol extract of Comparative Example 10 after being exposed to light for different days are as follows: Figure 5 As shown, A represents the total polysaccharide retention rate, B represents the total polyphenol retention rate, and C represents the total flavonoid retention rate. The results show that after the Matsutake eutectic microemulsion of Example 1 was observed under light conditions for 90 days, the retention rate of total polysaccharides was 97.20%, the retention rate of total polyphenols was 78%, and the retention rate of total flavonoids was 82.09%. After 90 days of observation under light conditions, the retention rate of total polysaccharides of the Matsutake water extract of Comparative Example 9 was 96.29%, the retention rate of total polyphenols was 68.87%, and the retention rate of total flavonoids was 40.51%; after 90 days of observation under light conditions, the retention rate of total polysaccharides of the Matsutake alcohol extract of Comparative Example 10 was 96.09%, the retention rate of total polyphenols was 65.65%, and the retention rate of total flavonoids was 38.94%. It can be seen that the stability of the low eutectic microemulsion of Matsutake is better, indicating that the active ingredients of Matsutake extracted by the low eutectic solvent microemulsion are encapsulated inside the microcapsule structure, which significantly improves the stability of the light-sensitive total polyphenols and total flavonoids.

[0183] Test Example 4

[0184] The in vitro transdermal absorption test was conducted using the eutectic microemulsions of Tricholoma matsutake (extracted with eutectic solvent microemulsion) of Examples 1-11 and the aqueous extract of Tricholoma matsutake (water extraction) of Comparative Example 9. The Franz diffusion cell method was used, and the ex vivo pig ear back skin was used as a carrier, which was fixed between the supply pool and the receiving pool of the diffusion cell. The diffusion cell was fixed in a transdermal absorption diffusion instrument, and the phosphate buffer was used as the receiving liquid. The temperature of the receiving pool was maintained at 32°C, and the magnetic son maintained a rotation speed of 200 r / min. 2 g of the eutectic microemulsions of Tricholoma matsutake of Examples 1-11 or the aqueous extract of Tricholoma matsutake of Comparative Example 9 (the aqueous extract of Tricholoma matsutake of Comparative Example 9 was concentrated to the same total polysaccharide concentration as that of Example 1) was added to the release pool, and three replicates were set. The skin was removed after 1, 2, 4, 6, 8, 12 and 24 hours, the skin surface was wiped clean with absorbent cotton dipped in water for injection, and the skin surface was blotted dry with filter paper. The skin was cut into pieces and placed in a 5 mL centrifuge tube. 1 mL of phosphate buffer was added for homogenization. The operation was repeated once. The two homogenates were combined and centrifuged to obtain the supernatant. The phenol-sulfuric acid method was used to detect the content of total polysaccharides in the skin, and the total polysaccharide retention per unit area in the skin was calculated.

[0185] After testing, after 24 hours, the total polysaccharide retention in the skin of the Tricholoma matsutake eutectic microemulsion of Examples 1-11 was 68.64-72.69 μg / cm 2 .

[0186] The total polysaccharide intradermal retention results of the pine twig eutectic microemulsion of Example 1 and the pine twig water extract of Comparative Example 9 at different times are as follows: Figure 6 As shown in the results, the total polysaccharide retention in the skin of Example 1 and Comparative Example 9 was not detected in the 1st and 2nd hours, but the total polysaccharide could be detected from the 4th hour. After 24 hours, the total polysaccharide retention in the skin of the pine eutectic microemulsion of Example 1 was 72.69 μg / cm 2 ) is the Matsutake water extract of Comparative Example 9 (46.98 μg / cm 2 ), indicating that the smaller particle size of the Tricholoma matsutake eutectic microemulsion of the present invention makes it easier for the total polysaccharides to enter the skin, indicating that the Tricholoma matsutake eutectic microemulsion can improve the bioavailability of the total polysaccharides.

[0187] Test Example 5

[0188] The DPPH free radical scavenging experiment was carried out on the pine twig eutectic microemulsion (low eutectic solvent microemulsion extraction) of Examples 1-11, the pine twig water extract (water extraction) of Comparative Example 9, and the pine twig alcohol extract (alcohol extraction) of Comparative Example 10. Take 1 mg of DPPH and dissolve it in about 20 mL of solvent anhydrous ethanol, ultrasonicate for 5 min, shake it thoroughly, prepare a DPPH solution, and store it in the dark. The liquids of the examples and comparative examples were prepared into test solutions with corresponding concentrations (2, 4, 5, 6, 8, 10 mg / mL).

[0189] A0 value detection: Take 2 mL of DPPH solution and mix it thoroughly with 1 mL of anhydrous ethanol, and then measure the absorbance value A0 at 519 nm.

[0190] A value detection: Take 2 mL of DPPH solution and mix it thoroughly with 1 mL of the test solution of the embodiment or comparative example, and measure the absorbance value A at 519 nm. And calculate the DPPH free radical scavenging rate according to the following formula:

[0191]

[0192] After measurement, when the concentration was 10 mg / mL, the scavenging rate of the Tricholoma matsutake eutectic microemulsion of Examples 1-11 on DPPH free radicals was 80.36%~84.76%.

[0193] The DPPH radical scavenging results of the pine twig eutectic microemulsion of Example 1, the pine twig water extract of Comparative Example 9, and the pine twig alcohol extract of Comparative Example 10 are as follows: Figure 7 As shown, the results show that the antioxidant efficacy of the pine twig eutectic microemulsion of Example 1, the pine twig water extract of Comparative Example 9 and the pine twig alcohol extract of Comparative Example 10 increases with the increase of concentration within the concentration range of 2-10 mg / mL. When the concentration is 10 mg / mL, the DPPH radical scavenging rate of the pine twig eutectic microemulsion of Example 1 is 84.76%, the DPPH radical scavenging rate of the pine twig water extract of Comparative Example 9 is 55.84%, and the DPPH radical scavenging rate of the pine twig alcohol extract of Comparative Example 10 is 40.68%, indicating that the antioxidant capacity of the pine twig eutectic microemulsion of the present invention within the concentration range of 2-10 mg / mL is stronger than that of the pine twig solution extracted with traditional solvents (water, alcohol).

[0194] Test Example 6

[0195] Hydroxyl radical scavenging experiments were performed on the pine twig eutectic microemulsions (extracted with eutectic solvent microemulsions) of Examples 1-11, the pine twig water extract (water extraction) of Comparative Example 9, and the pine twig alcohol extract (alcohol extraction) of Comparative Example 10. The liquids of the examples and comparative examples were prepared into test solutions of corresponding concentrations (0.45, 0.89, 1.79, 3.57, 7.14 mg / mL), and the reaction system was prepared according to the instructions of the hydroxyl radical detection kit (Nanjing Jiancheng Bioengineering Research Institute Co., Ltd.) and the amount of each reagent added in Table 3, mixed well, and 3 replicate wells were set for each concentration.

[0196] Table 3

[0197]

[0198] After mixing and placing at room temperature for 20 minutes, 200 μL of each tube was transferred to a 96-well plate and the absorbance of each tube was detected at a wavelength of 550 nm. The calculation formula for the hydroxyl radical scavenging rate is as follows:

[0199]

[0200] After measurement, when the concentration was 7.14 mg / mL, the scavenging rate of the Tricholoma matsutake eutectic microemulsion of Examples 1-11 on hydroxyl radicals was 75.05%~77.16%.

[0201] The hydroxyl radical scavenging results of the pine twig eutectic microemulsion of Example 1, the pine twig water extract of Comparative Example 9, and the pine twig alcohol extract of Comparative Example 10 are as follows: Figure 8 As shown, the results show that the antioxidant efficacy of the pine twig eutectic microemulsion of Example 1, the pine twig water extract of Comparative Example 9 and the pine twig alcohol extract of Comparative Example 10 increases with the increase of the dilution concentration in the concentration range of 0.45~7.14 mg / mL. When the dilution is 7.14 mg / mL, the hydroxyl radical scavenging rate of the pine twig eutectic microemulsion of Example 1 is 77.16%, the hydroxyl radical scavenging rate of the pine twig water extract of Comparative Example 9 is 73.23%, and the hydroxyl radical scavenging rate of the pine twig alcohol extract of Comparative Example 10 is 64.30%, indicating that the antioxidant capacity of the pine twig eutectic microemulsion is stronger than that of the pine twig solution extracted with traditional solvents (water, alcohol).

[0202] Test Example 7

[0203] 1. Methods

[0204] The pine twig eutectic microemulsions (extraction with eutectic solvent microemulsions) of Examples 1-11, the pine twig water extract (water extraction) of Comparative Example 9, and the pine twig alcohol extract (alcohol extraction) of Comparative Example 10 were selected for cytotoxicity tests, and the cytotoxicity was measured by the relative activity of human immortalized keratinocytes (HaCaT). The specific experimental scheme is as follows:

[0205] (1) Cell seeding: 1×10 4 Cells were seeded into 96-well plates at 100 cells / well and incubated overnight in an incubator (37°C, 5% CO2).

[0206] (2) Experimental grouping: The experiment was divided into a zero adjustment group, a solvent control group, and a sample group. In the sample group, each sample was set with 8 concentration gradients, and 3 replicate wells were set under each concentration gradient.

[0207] (3) Solution preparation: The concentration gradient of the sample group is shown in Table 4 below.

[0208] Table 4

[0209]

[0210] (4) Administration: Administration was performed when the cell plating rate in the 96-well plate reached 40%-60%. In the solvent control group, 200 μL of culture medium containing 10% PBS was added to each well; in the sample group, 200 μL of culture medium containing the corresponding concentration of sample was added to each well; in the zero adjustment group, no cells were inoculated, and only 200 μL of cell culture medium was added. After administration, the 96-well plate was placed in an incubator (37°C, 5% CO2) for incubation.

[0211] (5) Detection: After incubating the cells for 24 h, discard the supernatant, add MTT working solution (0.5 mg / mL), and incubate at 37°C in the dark for 4 h. After incubation, discard the supernatant, add 100 µL DMSO to each well, and read the OD value at 490 nm.

[0212] (6) Calculation of cell viability: Calculate according to the following formula:

[0213]

[0214] (7) Half inhibitory concentration (IC 50 ) Calculation: Based on the cell viability results, use Excel to fit and draw the dose-effect curve, and calculate the half inhibitory concentration IC based on the curve 50 .

[0215] 2. Results

[0216] After testing, the eutectic microemulsions of Tricholoma matsutake of Examples 1-11 showed no cytotoxicity at a concentration of 0.30% to 0.32% (V / V). The eutectic microemulsion of Tricholoma matsutake of Example 1 showed no cytotoxicity at a concentration of 0.30% (V / V), while the water extract of Tricholoma matsutake of Comparative Example 9 showed no cytotoxicity at a concentration of 0.02% (V / V), and the alcohol extract of Tricholoma matsutake of Comparative Example 10 showed no cytotoxicity at a concentration of 0.02% (V / V). The cytotoxicity of the eutectic microemulsions of Tricholoma matsutake of the examples was lower than that of the water extract of Tricholoma matsutake of Comparative Example 9 and the alcohol extract of Tricholoma matsutake of Comparative Example 10.

[0217] The cytotoxicity test results of Example 1 and Comparative Examples 9 and 10 are shown in Table 5 below.

[0218] Table 5

[0219]

[0220] Test Example 8

[0221] 1. Methods

[0222] ROS detection was performed on the pine twig eutectic microemulsion (extraction with eutectic solvent microemulsion) of Examples 1-11, the pine twig water extract (water extraction) of Comparative Example 9, and the pine twig alcohol extract (alcohol extraction) of Comparative Example 10, and the antioxidant effect was evaluated based on the change in ROS content. The specific scheme is as follows:

[0223] (1) Cell seeding: 1×10 5 Cells were seeded into 24-well plates at 100 cells / well and incubated overnight in an incubator (37°C, 5% CO2).

[0224] (2) Experimental setup: Test according to the experiment set in Table 6 below.

[0225] Table 6

[0226]

[0227] Note: VC in the table stands for vitamin C and VE stands for vitamin E.

[0228] (3) Drug administration: After culturing in an incubator (37°C, 5% CO2) for 24 h, add drugs according to Table 6 and continue culturing for another 24 h.

[0229] (4) Modeling: The cells were washed twice with PBS and untreated cells were used as blank controls. The other groups were stimulated with 10 mW blue light for 24 h according to the conditions in the table, with the VC+VE group as the positive control.

[0230] (5) ROS content detection: The 2,7-dichlorofluorescein diacetate (DCFH-DA) probe stock solution was diluted with serum-free culture medium at a ratio of 1:1000 to a final concentration of 10 μmol / L. 500 μL of the diluted DCFH-DA probe was added to each well and incubated in a 37°C cell culture incubator. After 30 min, the cells were washed 4 times with serum-free DMEM culture medium to fully remove the probe that did not enter the cells. Using a fluorescence microscope, the cells were observed and photographed at an excitation wavelength of 488 nm, and the ROS fluorescence image was recorded.

[0231] (6) Analyze the relative integrated optical density (IOD) value of each group of photos and calculate the relative reduction rate of ROS content according to the following formula:

[0232] .

[0233] 2. Results

[0234] After measurement, compared with the NC group, the fluorescence signals of the PC group and the experimental group were significantly reduced, among which the fluorescence signal of the embodiment group was significantly reduced to a higher degree than that of comparative examples 9 and 10. The relative reduction rate of ROS content in the eutectic microemulsions of Tricholoma matsutake of Examples 1-11 with a concentration of 0.020% was 33.61-35.37%, among which the relative reduction rate of ROS content in the eutectic microemulsion of Tricholoma matsutake of Example 1 was 35.37%. The relative reduction rate of ROS content in the water extract of Tricholoma matsutake of comparative example 9 was 25.61%, and the relative reduction rate of ROS content in the alcohol extract of Tricholoma matsutake of comparative example 10 was 20.73%. It shows that the antioxidant effect of the eutectic microemulsion of Tricholoma matsutake of the present invention at a concentration of 0.020% (V / V) is stronger than that of the Tricholoma matsutake solution extracted by traditional solvents (water, alcohol).

[0235] The fluorescence images of Example 1, Comparative Example 9 and Comparative Example 10 in the control group and the experimental group are as follows: Fig. 9 As shown, the relative IOD value bar graphs of Example 1, Comparative Example 9, and Comparative Example 10 in the control group and the experimental group are shown in Fig.10 shown.

[0236] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the essence and scope of the technical solution of the present invention.

Claims

1. A Tricholoma matsutake eutectic microemulsion, characterized in that: The dry powder of Tricholoma matsutake and the microemulsion of low eutectic solvent are mixed, stirred, refluxed and extracted at 30-60°C, cooled to room temperature and filtered, and the filtrate is prepared; The deep eutectic solvent microemulsion comprises the following components in weight percentage: 1-10% of the deep eutectic solvent, 20-40% of the surfactant, 10-15% of the co-surfactant, and the balance is water; The low eutectic solvent is prepared from a hydrogen bond donor and a hydrogen bond acceptor, and the molar ratio of the hydrogen bond acceptor to the hydrogen bond donor is 1:(1-3); The hydrogen bond acceptor is lauric acid, and the hydrogen bond donor is capric acid; or the hydrogen bond acceptor is betaine, and the hydrogen bond donor is propionic acid; or the hydrogen bond acceptor is choline chloride, and the hydrogen bond donor is acetic acid; or the hydrogen bond acceptor is lauric acid, and the hydrogen bond donor is valeric acid; or the hydrogen bond acceptor is lauric acid, and the hydrogen bond donor is pyruvic acid; or the hydrogen bond acceptor is coconut oil, and the hydrogen bond donor is palm kernel oil; or the hydrogen bond acceptor is coconut acid, and the hydrogen bond donor is babassu seed oil.

2. The Tricholoma matsutake eutectic microemulsion according to claim 1, characterized in that The surfactant is one or more of Tween 20, Tween 60, Tween 80, Span 20, Span 60, PEG-20 hydrogenated castor oil, PEG-40 hydrogenated castor oil, and PEG-60 hydrogenated castor oil.

3. The Tricholoma matsutake eutectic microemulsion according to claim 1, characterized in that The co-surfactant is one or more of ethanol, 1,3-butylene glycol, dipropylene glycol, 1,2-propylene glycol, 1,3-propylene glycol, 1,2-hexanediol, and 1,2-pentanediol.

4. The Tricholoma matsutake eutectic microemulsion according to claim 1, characterized in that The preparation method of the low eutectic solvent microemulsion is: uniformly mix the low eutectic solvent, the surfactant and the co-surfactant, add water and stir until the system is clear and transparent, so as to obtain the low eutectic solvent microemulsion.

5. The Tricholoma matsutake eutectic microemulsion according to claim 4, characterized in that The uniform mixing is performed in an environment with a temperature of 25-40°C.

6. A method for preparing the Tricholoma matsutake eutectic microemulsion according to any one of claims 1 to 5, characterized in that: The Tricholoma matsutake dry powder is mixed with the low eutectic solvent microemulsion, stirred, extracted under reflux at 30-60° C., cooled to room temperature and filtered, and the filtrate is the Tricholoma matsutake low eutectic microemulsion.

7. The preparation method according to claim 6, characterized in that: The mass volume ratio of the Tricholoma matsutake dry powder to the low eutectic solvent microemulsion is 1g: (20-40)mL.

8. The preparation method according to claim 6, characterized in that: The stirring speed is 100-300 r / min; And / or, the extraction time is 2 to 4 hours.

9. Use of the Tricholoma matsutake eutectic microemulsion according to any one of claims 1 to 5 in the preparation of skin care products.

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