A supramolecular cypress extract, a preparation method thereof and application thereof

Abstract

The present application relates to the technical field of plant extraction, and particularly relates to a supramolecular biota extract, a preparation method and application thereof, the preparation method comprising the following steps: mixing L-carnitine taurine ionic liquid and biota leaves, and performing soaking treatment to obtain a liquid; performing ultrasonic extraction treatment on the liquid to obtain the supramolecular biota extract. The present application comprehensively uses soaking treatment and ultrasonic extraction treatment to extract biota leaves, and the L-carnitine taurine ionic liquid used after the extraction process does not need to be separated from the biota leaf extract, and can form a new efficacy substance, the supramolecular biota extract, with the extracted biota leaf extract. The supramolecular biota extract has good penetration efficacy, cell and biological compatibility, and antioxidant property, and has the characteristics of high bioavailability, stable property, and the like when applied to cosmetics.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of plant extraction, in particular to a supramolecular cypress extract and a preparation method and application thereof. BACKGROUND

[0002] Cypress leaf, also known as cypress leaf, cluster cypress leaf, taxodium, and yunpi cypress, is the dried tender branch tips and leaves of Taxodium distichum. It is found in the Shengfa recipe of Jin·Ge Hong's Elbow Post Prescription that cypress leaf has the effect of treating hair loss. The main effective components of cypress leaf are quercetin and rutin, which can activate human hair matrix cells and promote blood circulation, revive hair follicles with declining hair growth ability, and supplement nutrients after promoting blood circulation to play the effect of hair growth. However, the washing and hair care products generally have the disadvantages of short residence time, poor water solubility, and poor drug dispersibility of effective components such as quercetin and rutin, which cannot enter the hair follicles, sweat glands and other parts for effective absorption at regular intervals and in a quantitative manner.

[0003] In addition, the solvents commonly used in the extraction of cypress leaf at present are water and ethanol, which have poor extraction effect on cypress leaf and low bioavailability of the extract.

[0004] Therefore, the prior art still needs to be improved and developed. SUMMARY

[0005] In view of the shortcomings of the prior art, the purpose of the present application is to provide a supramolecular cypress extract and a preparation method and application thereof, aiming to solve the problems of poor extraction effect of the existing cypress leaf extraction method and low bioavailability of the extract.

[0006] The technical scheme of the present application is as follows:

[0007] A preparation method of a supramolecular cypress extract, comprising the steps of:

[0008] providing a l-carnitine taurine ionic liquid;

[0009] mixing the l-carnitine taurine ionic liquid with cypress leaf and performing soaking treatment to obtain a feed liquid;

[0010] performing ultrasonic extraction treatment on the feed liquid to obtain a supramolecular cypress extract.

[0011] The preparation method of the supramolecular cypress extract, wherein the step of mixing the l-carnitine taurine ionic liquid with cypress leaf specifically comprises:

[0012] adding deionized water to the l-carnitine taurine ionic liquid to dilute it to obtain an l-carnitine taurine ionic liquid aqueous solution, and then mixing the l-carnitine taurine ionic liquid aqueous solution with cypress leaf.

[0013] The method for preparing the supramolecular arborvitae extract, wherein the volume of the L-carnitine taurine ionic liquid accounts for 2% to 8% of the volume of the L-carnitine taurine ionic liquid aqueous solution.

[0014] The method for preparing the supramolecular arborvitae extract, wherein the material-to-liquid ratio between the mass of the arborvitae leaves and the volume of the L-carnitine taurine ionic liquid aqueous solution is 1 g:(10~30) mL.

[0015] The method for preparing the supramolecular Platycladus orientalis extract, wherein the method for preparing the L-carnitine taurine ionic liquid includes the following steps:

[0016] Taurine and L-carnitine were dissolved in water to obtain taurine solution and L-carnitine solution, respectively.

[0017] The L-carnitine solution was added to the taurine solution, and after the reaction, vacuum distillation was performed to obtain an L-carnitine taurine ionic liquid.

[0018] The method for preparing the supramolecular arborvitae extract, wherein the molar ratio of L-carnitine to taurine is (1~3):(3~1).

[0019] The method for preparing the supramolecular arborvitae extract, wherein the soaking temperature is 20-30℃ and the soaking time is 1-3h.

[0020] The method for preparing supramolecular arborvitae extract includes the following steps: the ultrasonic input power for ultrasonic extraction is 300W, the ultrasonic frequency for ultrasonic extraction is 40kHz, the ultrasonic extraction temperature is 30-70℃, and the ultrasonic extraction time is 10-50min.

[0021] A supramolecular arborvitae extract, prepared using the same method as the supramolecular arborvitae extract.

[0022] Application of a supramolecular arborvitae extract in the preparation of cosmetics.

[0023] Beneficial effects: the supramolecular cypress extract provided by the application, the left-handed carnitine taurine ionic liquid and the flavonoid effective component in cypress leaves are combined through hydrogen bond and van der waals interaction, these interactions can improve the combination efficiency of the flavonoid effective component and the ionic liquid, greatly improving the extraction efficiency of the flavonoid effective component in cypress leaves. At the same time, the cypress leaves are extracted by comprehensively using soaking treatment and ultrasonic treatment, the soaking treatment is helpful for the full contact of the ionic liquid and the cypress leaves, the left-handed carnitine taurine ionic liquid can cause damage to the plant cell wall and increase the cell permeability, the ultrasonic treatment can further promote the cell wall permeability or cell rupture, accelerate the active substances in the cells to enter the extraction liquid, and the left-handed carnitine taurine ionic liquid used after the extraction process does not need to be separated, and can be combined with the extracted cypress leaf extract to form a new efficacy substance, the supramolecular cypress extract. The supramolecular cypress extract has good penetration efficacy, cell and biological compatibility, antioxidant property, and has the characteristics of high bioavailability, stable properties, etc. when applied to cosmetics. BRIEF DESCRIPTION OF DRAWINGS

[0024] Figure 1 It is a process flow diagram for the preparation method of the supramolecular cypress extract of the application;

[0025] Figure 2 It is a SEM diagram of example 1 and comparative example 1 and comparative example 2;

[0026] Figure 3 It is a penetration test diagram of example 1 and comparative example 1;

[0027] Figure 4 It is a cytotoxicity test diagram of the supramolecular cypress extract in example 1;

[0028] Figure 5 It is an ABTS + free radical scavenging curve diagram of the supramolecular cypress extract in example 1;

[0029] Figure 6 It is an IL-1ɑ content detection diagram of human keratinocytes treated with the supramolecular cypress extract in example 1;

[0030] Figure 7 It is a 6h oil average change rate result diagram of example 1 and comparative example 1;

[0031] Figure 8 It is a VEGF content test diagram of example 1 and comparative example 1. DETAILED DESCRIPTION

[0032] The application provides a supramolecular biota extract, a preparation method and application thereof. To make the purpose, technical scheme and effect of the application clearer and more explicit, the application is further described in detail below. It should be understood that the specific embodiments described herein are only used to explain the application and not to limit the application.

[0033] Those skilled in the art can understand that, unless otherwise defined, all terms (including technical terms and scientific terms) used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. It should also be understood that terms such as those defined in a general dictionary should be understood in the context of the prior art and should not be interpreted in an idealized or overly formal sense unless specifically defined as such.

[0034] As shown in Figure 1 The application provides a preparation method of a supramolecular biota extract, comprising the steps of:

[0035] Step S10: providing a L-carnitine taurine ionic liquid;

[0036] Step S20: mixing the L-carnitine taurine ionic liquid with biota leaves, and performing soaking treatment to obtain a feed liquid;

[0037] Step S30: performing ultrasonic extraction treatment on the feed liquid to obtain a supramolecular biota extract.

[0038] In this embodiment, the ionic liquid, as a green and environmentally friendly solvent, has the characteristics of low melting point and high polarity, can dissolve various organic and inorganic substances, has a high extraction efficiency on plants, and can reduce the pollution of organic solvents to the environment and the harm to humans. In addition, the addition of active substances in some ionic liquids with good biocompatibility can combine with the effective components in the biota liquid to play a synergistic effect, and the ionic liquid also has a good penetration effect.

[0039] Specifically, the step of mixing the L-carnitine taurine ionic liquid with the biota leaves specifically comprises: adding deionized water to the L-carnitine taurine ionic liquid for dilution to obtain an L-carnitine taurine ionic liquid aqueous solution, and then mixing the L-carnitine taurine ionic liquid aqueous solution with the biota leaves for soaking treatment to obtain a feed liquid. Soaking the biota leaves in the L-carnitine taurine ionic liquid aqueous solution can fully soak the extract, improve the extraction efficiency, and at the same time, the L-carnitine taurine ionic liquid can destroy the cell wall, which is conducive to the full extraction of active ingredients.

[0040] Further, the extraction rate of the plant active ingredient is closely related to the interaction between the active ingredient and the solvent. The flavonoids contain many active hydrogens and unsaturated bonds. The L-carnitine taurine ionic liquid interacts with the flavonoids through hydrogen bond interaction and van der Waals interaction. These interactions can improve the binding efficiency of the flavonoids and the ionic liquid, and greatly improve the extraction rate of the flavonoids in the cypress leaves.

[0041] In addition, the cypress leaves are extracted by comprehensively using soaking and ultrasonic. The soaking treatment is helpful for the full contact of the ionic liquid with the cypress leaves. The ultrasonic treatment can promote the cell wall permeability or cell rupture, accelerate the active substances in the cells into the extraction liquid, and improve the extraction efficiency.

[0042] Therefore, the extraction method using the L-carnitine taurine ionic liquid as the extraction agent has high feasibility and high extraction efficiency, can extract and retain the active ingredients in the cypress leaves to the greatest extent, and improves the utilization efficiency of biomass resources. After extraction, the L-carnitine taurine ionic liquid does not need to be separated, and can be combined with the extracted cypress leaf extract to form a new efficacy product, to obtain a supramolecular cypress extraction liquid.

[0043] Specifically, the L-carnitine has good anti-apoptosis activity, can stimulate the proliferation of keratinocytes, and inhibit the apoptosis of hair matrix keratinocytes in the growth phase, so as to promote hair growth. In addition, the L-carnitine also has skin moisturizing effect. The taurine is one of the free amino acids with the largest content in the human body, can effectively regulate the normal physiological activities of the human body, has various physiological and chemical activities, is closely related to cell proliferation and apoptosis, and has skin antioxidant and anti-inflammatory effects. The flavonoid active substances in the cypress leaves can activate human hair matrix cells and promote blood circulation, and promote hair growth. The use of the L-carnitine taurine ionic liquid as the extraction solvent makes the newly prepared supramolecular cypress extraction liquid contain the L-carnitine taurine ionic liquid, which can promote the transdermal penetration effect of the flavonoid active substances. In addition, the supramolecular cypress extraction liquid has the effects of the taurine and the L-carnitine in the ionic liquid, and the effects of the flavonoid active substances in the cypress leaves, can regulate the development, growth and apoptosis of hair cells, has the effects of hair growth, hair care, scalp oil control and scalp soothing, and has the characteristics of high penetration, high bioavailability and stable properties when applied to cosmetics.

[0044] In some embodiments, the volume of the L-carnitine taurine ionic liquid accounts for 2% to 8% of the volume of the L-carnitine taurine ionic liquid aqueous solution. Controlling the content of the L-carnitine taurine ionic liquid in the L-carnitine taurine ionic liquid aqueous solution can efficiently extract the active substances in the cypress leaves during the extraction of the cypress leaves, and improve the extraction effect.

[0045] In a preferred embodiment, the extracting agent is a 5% aqueous solution of L-carnitine taurine ionic liquid, and the extraction of the cypress leaves is optimal, the extraction efficiency is high, the active ingredients in the cypress leaves can be extracted and retained to the maximum extent, and the utilization efficiency of biomass resources is improved.

[0046] In some embodiments, the method for preparing the L-carnitine taurine ionic liquid in the step S10 comprises the following steps:

[0047] Step S11: Dissolving taurine and L-carnitine in water respectively to obtain a taurine solution and an L-carnitine solution;

[0048] Step S12: Adding the L-carnitine solution into the taurine solution, and performing vacuum distillation after reaction to obtain the L-carnitine taurine ionic liquid.

[0049] Specifically, the two monomers of taurine and L-carnitine can be prepared into ionic liquid by the above method for preparing the L-carnitine taurine ionic liquid, so that the extraction efficiency can be improved when the cypress leaves are extracted by the ionic liquid, and the obtained extract does not need to separate the ionic liquid, and the transdermal penetration effect of the flavonoid active substances can be promoted.

[0050] In some embodiments, in the step S12, the L-carnitine solution is added into the taurine solution, and the reaction is performed at 25°C for 8 hours. After the reaction is completed, the aqueous solution is removed by vacuum distillation at 60°C to obtain the L-carnitine taurine ionic liquid.

[0051] In some embodiments, the molar ratio of the L-carnitine to the taurine is (1-3):(3-1). When the L-carnitine taurine ionic liquid is prepared according to the molar ratio, the reaction can be more complete, and the preparation efficiency is high.

[0052] In some embodiments, the molar ratio of the L-carnitine to the taurine is 1:1. The raw materials can be better reacted completely, the raw materials can be saved, and the cost can be reduced.

[0053] In some embodiments, the ratio of the mass of the cypress leaves to the volume of the aqueous solution of the L-carnitine taurine ionic liquid is 1g:(10-30)mL. According to the ratio, the aqueous solution of the L-carnitine taurine ionic liquid can efficiently extract the flavonoid active ingredients in the cypress leaves, and the extraction rate is high.

[0054] Specifically, the flavonoids in the cypress leaves can be extracted with high efficiency and high feasibility by the ratio of 1g: (10-30)mL between the mass of the cypress leaves and the volume of the aqueous solution of the L-carnitine taurine ionic liquid, so that the active ingredients in the cypress leaves can be extracted and reserved to the maximum extent, and the utilization efficiency of biomass resources is improved.

[0055] In a preferred embodiment, the ratio between the mass of the cypress leaves and the volume of the aqueous solution of the L-carnitine taurine ionic liquid is 1g:20mL, so that the flavonoids in the cypress leaves can be dissolved in the aqueous solution of the ionic liquid to the maximum extent, and the extraction rate is improved.

[0056] In some embodiments, the temperature of the soaking treatment is 20-30℃, and the time of the soaking treatment is 1-3h, so that the cypress leaves can be fully soaked, the overall extraction time is shortened, and the efficiency is improved.

[0057] In a preferred embodiment, the temperature of the soaking treatment is 25℃, and the time of the soaking treatment is 2h, so that the extraction time of the ionic liquid on the cypress leaves is shortened, and the active ingredients are not damaged.

[0058] In some embodiments, the ultrasonic input power of the ultrasonic extraction treatment is 300W, the ultrasonic frequency of the ultrasonic extraction treatment is 40kHz, the temperature of the ultrasonic extraction treatment is 30-70℃, and the time of the ultrasonic extraction treatment is 10-50min. By using the ultrasonic extraction treatment under the above conditions, the soaked material liquid is further extracted, the dissolution of the ionic liquid on the flavonoids is improved, and the extraction rate of the ionic liquid on the cypress leaves is improved.

[0059] In a preferred embodiment, the ultrasonic input power of the ultrasonic extraction treatment is 300W, the ultrasonic frequency of the ultrasonic extraction treatment is 40kHz, the temperature of the ultrasonic extraction treatment is 50℃, and the time of the ultrasonic extraction treatment is 30min.

[0060] In some embodiments, after the ultrasonic extraction treatment of the material liquid in the step S30, the material liquid is further subjected to a filtration treatment, a carbon powder decolorization treatment and a concentration treatment. The solid in the material liquid after the ultrasonic extraction treatment is removed by filtration, the color transparent liquid is obtained by the carbon powder decolorization treatment, and finally the concentration of the supramolecular cypress extraction liquid is improved by the concentration treatment.

[0061] In addition, the application further provides a supramolecular cypress extraction liquid prepared by the preparation method.

[0062] In the present embodiment, the supramolecular thuja extract prepared by the above preparation method not only has the effects of taurine and L-carnitine in the ionic liquid, but also has the effect of flavonoids active substances in thuja leaves, and the percutaneous penetration effect of the flavonoids active substances is promoted by the ionic liquid component. The supramolecular thuja extract has good penetration efficacy, cell and biological compatibility, and antioxidant properties.

[0063] In addition, the present application also provides a use of the supramolecular thuja extract in the preparation of cosmetics.

[0064] In some embodiments, the supramolecular thuja extract has good penetration efficacy, cell and biological compatibility, and antioxidant properties, and when applied in cosmetics, has the effects of transdermal oil control and transdermal soothing, and the supramolecular thuja extract has high bioavailability, stable properties, and the like.

[0065] The following examples are further provided to illustrate the present application in detail. It should also be understood that the following examples are only used to further illustrate the present application, and cannot be understood as limiting the scope of protection of the present application. Any non-essential improvements and adjustments made by those skilled in the art based on the above content of the present application shall fall within the scope of protection of the present application.

[0066] Example 1

[0067] A certain amount of taurine was dissolved in water at room temperature, and then an aqueous solution of L-carnitine was added dropwise to the solution containing taurine, the molar ratio of L-carnitine to taurine being 1:1, and the reaction was carried out at 25°C for 8 hours. After the reaction was completed, the aqueous solution was removed by vacuum distillation at 60°C to obtain L-carnitine taurine ionic liquid.

[0068] The dry thuja leaf powder was mixed with an aqueous solution containing 5% L-carnitine taurine ionic liquid at a solid-liquid ratio (g:mL) of 1:20, and soaked at 25°C for 2 hours. The soaked solid-liquid mixture was subjected to ultrasonic extraction at an ultrasonic input power of 300W, an ultrasonic frequency of 40kHz, and a temperature of 50°C for 30 minutes. After filtration, carbon powder decolorization, and concentration, a supramolecular thuja extract was obtained.

[0069] Example 2

[0070] The dry thuja leaf powder was mixed with an aqueous solution containing 5% L-carnitine taurine ionic liquid at a solid-liquid ratio (g:mL) of 1:10, and soaked at 25°C for 2 hours. The soaked solid-liquid mixture was subjected to ultrasonic extraction at an ultrasonic input power of 300W, an ultrasonic frequency of 40kHz, and a temperature of 60°C for 40 minutes. After filtration, carbon powder decolorization, and concentration, a supramolecular thuja extract was obtained.

[0071] Example 3

[0072] The dry cypress leaf powder was mixed with a 5% aqueous solution of L-carnitine taurine ionic liquid at a solid-liquid ratio (g:mL) of 1:30, and soaked at 25°C for 1 h. The soaked solution was subjected to ultrasonic extraction at an ultrasonic input power of 300 W, an ultrasonic frequency of 40 kHz, and a temperature of 40°C for 20 min. After filtration, carbon powder decolorization, and concentration, a supramolecular cypress extract was obtained.

[0073] Comparative Example 1

[0074] The dry cypress leaf powder was mixed with a 5% aqueous solution of commercially available 1,3-dimethylimidazole dimethyl phosphate ionic liquid at a solid-liquid ratio (g:mL) of 1:20, and soaked at 25°C for 2 h. The soaked solution was subjected to ultrasonic extraction at an ultrasonic input power of 300 W, an ultrasonic frequency of 40 kHz, and a temperature of 50°C for 30 min. After filtration, carbon powder decolorization, and concentration, a cypress extract was obtained.

[0075] Comparative Example 2

[0076] The dry cypress leaf powder was mixed with deionized water at a solid-liquid ratio (g:mL) of 1:20, and soaked at 25°C for 2 h. The soaked solution was subjected to ultrasonic extraction at an ultrasonic input power of 300 W, an ultrasonic frequency of 40 kHz, and a temperature of 50°C for 30 min. After filtration, carbon powder decolorization, and concentration, a cypress water extract was obtained.

[0077] The flavonoid content of the supramolecular cypress extract obtained in Examples 1-3, the cypress extract obtained in Comparative Example 1, and the cypress water extract obtained in Comparative Example 2 was detected, and the total flavonoid extraction yield was compared as shown in Table 1.

[0078]

[0079] As can be seen from Table 1, the use of an aqueous solution of L-carnitine taurine ionic liquid as a solvent has a higher extraction efficiency than the use of commercially available 1,3-dimethylimidazole dimethyl phosphate ionic liquid aqueous solution and pure ionic liquid aqueous solution as a solvent.

[0080] By comparing the total flavonoid extraction yields obtained by the various preparation methods, and considering the time and equipment costs, it can be seen that the scheme provided in Example 1 has a higher extraction efficiency.

[0081] Test:

[0082] The soaked solutions of Examples 1, Comparative Example 1, and Comparative Example 2 were filtered, dried, and solid powders were obtained. The solid powders were subjected to SEM testing to observe the integrity of the surface morphology.

[0083] The experimental results are shown in Table 1. Figure 2

[0084] (a) in FIG. 1 is an SEM image of the solid powder of Comparative Example 2, Figure 2 (b) in FIG. 1 is an SEM image of the solid powder of Comparative Example 1, Figure 2 (c) in FIG. 1 is an SEM image of the solid powder of Example 1. It can be seen from the figure that the surface of the material in Comparative Example 2 is relatively complete and is not obviously damaged; the surface of the material in Comparative Example 1 is damaged and delamination occurs; the surface of the material in Example 1 is obviously damaged and the material is obviously delaminated and forms a honeycomb shape. It is shown that the ionic liquid provided by the application can cause relatively obvious damage to the plant morphology in extraction, which is beneficial to the dissolution of active ingredients, thereby improving the extraction rate and extraction efficiency. Figure 2 Performance test:

[0085] (I) Transdermal effect test:

[0086] The transdermal effect test was performed on the supermolecular thuja extract provided by Example 1 after dilution to the same total flavonoid content as Comparative Example 1, and the thuja extract provided by Comparative Example 1. The specific test method is as follows:

[0087] 1. The back skin of GF Kunming mice was used, the subcutaneous fat layer and connective tissue were carefully peeled off, and the skin was washed with physiological saline and placed in physiological saline for standby.

[0088] 2. The Franz cell method was used for transdermal experiment. The exposed mouse skin area in the diffusion cell in the Franz diffusion device was 1.13 cm 2 , and the receiving chamber volume was 15 mL.

[0089] 3. 1.0 mL of the test liquid was taken for the skin surface, 15 mL of commercially available PBS buffer salt solution (pH≈7.4) was added to the receiving pool, and the receiving pool was placed in a 32±0.5℃ constant temperature water bath, and the stirring speed was 350 rad / min.

[0090] 4. Subcutaneous permeation test: 2 mL of receiving liquid was taken at different time points, 3 parallel samples were taken at each time point, 2 mL of receiving liquid was supplemented into the receiving chamber immediately after sampling, and the receiving liquid taken at different time points was filtered through a 0.22 μm microporous filter membrane, and the concentration of the receiving liquid was detected by liquid phase detection.

[0091] The transdermal effect test results show that:

[0092] From

[0093] Figure 3 ​It can be seen that under the same experimental conditions, the permeation per unit area of ​​the supramolecular Platycladus orientalis extract provided in Example 1 and the Platycladus orientalis extract provided in Comparative Example 1 both increased continuously over time. Among them, the permeation per unit area of ​​the supramolecular Platycladus orientalis extract was 1222.79 μg / cm³. 2 The permeability per unit area of ​​the Platycladus orientalis extract was 491.46 μg / cm². 2 Experiments show that the supramolecular extract of Platycladus orientalis has a significantly better permeation effect than Platycladus orientalis extract, and the supramolecular extract of Platycladus orientalis has a better permeation effect.

[0094] (II) Cytotoxicity test:

[0095] 1. Using 1.5x10 4 Seed cells at a density of 100 μL per well into a 96-well plate and incubate for 24 h.

[0096] 2. After the cells adhered to the culture medium for 24 hours, discard the culture medium and add 100 μL of the supramolecular arborvitae extract from Example 1 to each well to prepare the test solution. Set up a blank group and a zeroing group.

[0097] 3. After incubating the cells for 24 hours, add 10 μL of MTT solution to each well and incubate for 4 hours.

[0098] 4. Discard the culture medium, add 150 μL of DMSO to each well, shake for 10 min, and then measure the absorbance (OD) at 490 nm. 490 The results (nm) were compared with those of the control sample that was not treated with the test substance and the results were calculated.

[0099] Figure 4 This is a cytotoxicity test diagram of the supramolecular Platycladus orientalis extract from Example 1. Figure 4 It can be seen that the supramolecular arborvitae extract still has more than 90% cell viability at 0.625% (v / v), indicating that the supramolecular arborvitae extract of Example 1 has good cell compatibility.

[0100] (III) Patch Test:

[0101] The supramolecular arborvitae extract provided in Example 1 was used for patch testing. The specific testing method is as follows:

[0102] 1. Select qualified patch testing equipment and use the closed patch test method. Place 0.020 g to 0.025 g of the test substance in the patch testing equipment and apply it to the flexor side of the subject's forearm with low-allergenic adhesive tape.

[0103] 2. Remove the test substance after 24 hours, and observe the skin reaction at 0.5, 24 and 48 hours after removal. Record the results according to the skin reaction grading standard in the "Cosmetic Safety Technical Specifications" (2015 edition).

[0104] Table 2 is a statistical table of patch test results for Example 1. As can be seen from Table 2, among the 33 people who underwent closed patch test on human skin using the supramolecular arborvitae extract provided in Example 1, 0 cases showed adverse skin reactions.

[0105]

[0106] (iv) Repeated skin irritation tests:

[0107] Experimental Method: Approximately 24 hours before the experiment, the hair on both sides of the spine on the back of the test animal was shaved without damaging the epidermis. The shaved area was approximately 3cm × 3cm on both the left and right sides. 0.5mL of the sample supramolecular arborvitae extract was applied to a 2.5cm × 2.5cm area on the shaved skin on the left side, with the other side serving as a control. Application was performed once daily for 14 consecutive days. Starting from the second day, the hair was shaved before each application, and any remaining test substance was removed with warm water. Local skin reactions were observed and scored 1 hour later.

[0108] Table 3 is a statistical table of the results of multiple skin irritation tests of the supramolecular arborvitae extract provided in Example 1. As can be seen from Table 3, the average score of each animal per day in the multiple skin irritation tests of the undiluted supramolecular arborvitae extract on New Zealand rabbits was 0. According to the skin irritation intensity classification of the skin irritation test in the "Cosmetic Safety Technical Specifications" (2015 edition), it is non-irritating.

[0109]

[0110] (v) Antioxidant test:

[0111] Experimental Method: Based on the sample characteristics and recommended dosage, an appropriate mass concentration gradient was set up, and sample solutions were prepared separately using PBS buffer as the solvent. Sample tubes (A) were set up. s ), Sample background (A) b ), sample blank tube (A0), and sample tube (A1) for each test concentration of each sample. s Three parallel tubes are required, and three parallel tubes are also required for the sample blank tube (A0). In the sample tube (A... s ) and sample background (A b Add 0.2 mL of the same concentration of sample solution to each of the sample tubes (A1, A2, and A3), and add 0.2 mL of PBS buffer to the blank sample tube (A0). s Add 0.8 mL of ABTS to both the sample tube (A0) and the blank sample tube (A0). + Working solution, sample background (A) b Add 0.8 mL of PBS buffer. Incubate in the dark for 6 minutes. Transfer the solution from each reaction tube into a 1 cm cuvette and measure the absorbance at 734 nm.

[0112] ABTS + Free radical scavenging rate (%) X=(1−A) s -A b A0)×100%

[0113] Figure 5 The ABTS of the supramolecular arborvitae extract provided in Example 1 + Free radical scavenging curve, from Figure 5 It can be seen that supramolecular arborvitae extract has an effect on ABTS. + IC50 (half-maximum scavenging concentration) of free radicals 50 =0.31% (95% confidence interval IC) 50 The concentration of the supramolecular Platycladus orientalis extract (0.25%~0.38%) indicates that the extract has antioxidant activity at this concentration.

[0114] (vi) Cellular test for soothing efficacy:

[0115] 1. Using 1x10 5 Cells were seeded at a density of cells / well into 24-well plates and incubated in an incubator.

[0116] 2. Solution preparation: Prepare working solutions for the test substances according to the test protocol (Table 4);

[0117]

[0118] 3. Dosing: According to the test protocol in Table 4, dosing is done in groups, with 1 mL of sample added to each well, 3 replicates per group, and the reaction time is 24 h;

[0119] 4. UVB stimulation: Except for the BC control group, all other groups received radiation at 300 mJ / cm². 2 Human keratinocytes were irradiated with UVB, and then incubated for 24 hours after the stimulation ended.

[0120] 5. Collect cell supernatant and determine IL-1α content using an ELISA kit.

[0121] Figure 6 The graph shows the IL-LA content of human keratinocytes after treatment with the supramolecular arborvitae extract provided in Example 1. Compared with the BC group, the IL-LA secretion level of the NC group was significantly increased, indicating that the stimulation conditions in this experiment were effective. Compared with the NC group, the L-LA secretion level of the PC group was significantly decreased, indicating that the positive control group in this experiment was effective. Compared with the NC group, the L-LA content of the supramolecular arborvitae extract provided in Example 1 was significantly reduced at a concentration of 2.5%, with an inhibition rate of 79.72%. Therefore, the supramolecular arborvitae extract has a certain soothing effect at this concentration.

[0122] (Seven), oil control effect test:

[0123] (1) Before using the sample

[0124] 1. One week before the test, the subjects stop using cosmetics or external drugs, and the night before the test, the subjects do not wash their heads.

[0125] 2. The eligible subjects fill in the informed consent form.

[0126] 3. On the test day, the subjects clean their heads in the laboratory with a unified shampoo.

[0127] 4. The subjects rest in an environment with a temperature of 21±1℃ and a relative humidity of 50±10% RH for 25 minutes.

[0128] 5. Use the skin oil test instrument to collect head oil data, take pictures of the scalp, and fill out the questionnaire.

[0129] 6. Distribute the test samples and usage instructions.

[0130] (2) After using the sample for 6 hours

[0131] 1. Re-test the oil data of the hairline in the blank area and the sample area.

[0132] Figure 7 is the 6h oil average change rate result graph of the sample supramolecular Platycladus orientalis extract provided in Example 1 and the sample Platycladus orientalis extract provided in Comparative Example 1, the results show that: after using the supramolecular Platycladus orientalis extract for 6 hours in the test area of the subjects' scalp, compared with the blank area, the relative oil control rate is -55.66%, and after using the commercial Platycladus orientalis extract for 6 hours in the test area of the subjects' scalp, compared with the blank area, the relative oil control rate is -45.96%, indicating that the relative oil control rate of the supramolecular Platycladus orientalis extract is better than that of the Platycladus orientalis extract.

[0133] (Eight), anti-hair loss effect test:

[0134] 1. Inoculation: inoculate cells into a 6-well plate at a seeding density of 3×10 5 cells / well, and incubate in an incubator (37℃, 5% CO2) overnight.

[0135] 2. Liquid preparation: according to the following different test groups (Table 5), different concentrations of test substances are prepared.

[0136]

[0137] 3. H2O2 stimulation: according to the test group, when the cell plating rate in the 6-well plate reaches about 60%, H2O2 stimulation is performed for the groups with H2O2 stimulation, and the induction time is 1h.

[0138] 4. Drug administration: After the induction time, the drug was administered according to the test group, 2 mL per well, and 3 replicates per group. Incubate in a 37°C, 5% CO2 incubator for 23 hours.

[0139] 5. ELISA detection: After incubation, collect the cell culture supernatant in a centrifuge tube and detect and analyze according to the operation manual of the VEGF ELISA kit.

[0140] 6. Statistical analysis of results: Use GraphPad Prism to plot the results, which are expressed as Mean ± SD. Use t-test for statistical analysis between groups. The statistical analysis is two-tailed. P < 0.05 is considered to be significantly different, and P < 0.01 is considered to be extremely significantly different.

[0141] The test results are shown in Table 2: Figure 8 As shown in Table 2, compared with the control group, the VEGF promotion rate of the sample supramolecular Platycladus orientalis extract provided in Example 1 was 133.30% at a concentration of 0.625% (v / v), and the VEGF promotion rate of the sample Platycladus orientalis extract provided in Comparative Example 1 was 60.31% at a concentration of 0.625% (v / v). This indicates that the sample supramolecular Platycladus orientalis extract and the sample Platycladus orientalis extract can achieve the anti-alopecia effect by promoting the content of VEGF at this concentration, and the sample supramolecular Platycladus orientalis extract has a better anti-alopecia effect. It is speculated that the levomegacine in the levomegacine taurine ionic liquid has a certain anti-alopecia effect, and the ionic liquid has a penetration-enhancing effect on the flavonoid active ingredients in Platycladus orientalis in the transdermal test, so the anti-alopecia effect of the supramolecular Platycladus orientalis extract is better than that of the sample Platycladus orientalis extract.

[0142] (Nine), nine routine tests and pesticide residue tests:

[0143] 1. The sample supramolecular Platycladus orientalis extract provided in Example 1 was subjected to nine routine tests, including total bacterial count, total mold and yeast count, heat-resistant coliform group, Staphylococcus aureus, Pseudomonas aeruginosa, mercury, lead, arsenic, cadmium, and other items. The sample was tested, and the results of the tested items met the standard requirements, as shown in Table 6.

[0144]

[0145] 2. The sample supramolecular Platycladus orientalis extract provided in Example 1 was subjected to pesticide residue tests, including BHC (four isomers) and DDT (six isomers). The sample was tested, and the results of the tested items met the standard requirements, as shown in Table 7.

[0146]

[0147] In summary, the application provides a kind of supramolecular biota extract and its preparation method and application, the preparation method of the supramolecular biota extract includes the following steps: providing L-carnitine taurine ionic liquid;The L-carnitine taurine ionic liquid is mixed with biota leaf, and soaking treatment is carried out, to obtain liquor;The liquor is treated by ultrasonic extraction, to obtain supramolecular biota extract.The extraction efficiency of ionic liquid to flavonoids in biota leaf is closely related to the interaction between ionic liquid and flavonoids, and flavonoids contain many active hydrogen and unsaturated bonds, L-carnitine taurine ionic liquid interacts with flavonoids through hydrogen bond interaction and van der waals interaction, these interactions can improve the binding efficiency of flavonoids and ionic liquid, greatly improve the extraction efficiency of flavonoids in biota leaf.At the same time, the application comprehensively uses soaking treatment and ultrasonic extraction treatment to extract biota leaf, soaking treatment helps ionic liquid to fully contact with biota leaf, L-carnitine taurine ionic liquid can damage plant cell wall and increase cell permeability, ultrasonic treatment can further promote cell wall permeability or cell rupture, accelerate the active substances in cells into the extract, and the L-carnitine taurine ionic liquid used after extraction process does not need to be separated from biota extract, and can be combined with the extracted biota extract to form a new efficacy substance-supramolecular biota extract.The supramolecular biota extract has good penetration efficacy, cell and biological compatibility, antioxidant property, and when applied to cosmetics, has the characteristics of scalp oil control, scalp soothing, high bioavailability, stable properties and the like.

[0148] It should be understood that the application of the application is not limited to the above examples, and those skilled in the art can improve or change according to the above description, and all these improvements and changes should belong to the protection scope of the appended claims of the application.

Claims

1. A method for preparing a supramolecular Platycladus orientalis extract, characterized in that, Including the following steps: Provides L-carnitine taurine ionic liquid; The L-carnitine taurine ionic liquid was mixed with arborvitae leaves and soaked to obtain a liquid solution. The liquid material was subjected to ultrasonic extraction to obtain a supramolecular arborvitae extract; The soaking treatment temperature is 20-30℃, and the soaking treatment time is 1-3h; the ultrasonic input power of the ultrasonic extraction treatment is 300W, the ultrasonic frequency of the ultrasonic extraction treatment is 40kHz, the ultrasonic extraction treatment temperature is 30-70℃, and the ultrasonic extraction treatment time is 10-50min. The preparation method of the L-carnitine taurine ionic liquid includes the following steps: Taurine and L-carnitine were dissolved in water to obtain taurine solution and L-carnitine solution, respectively. The L-carnitine solution was added to the taurine solution, and after the reaction, vacuum distillation was carried out at 60°C to obtain L-carnitine taurine ionic liquid. The molar ratio of L-carnitine to taurine is (1~3):(3~1).

2. The method for preparing supramolecular Platycladus orientalis extract according to claim 1, characterized in that, The specific steps of mixing the L-carnitine taurine ionic liquid with Platycladus orientalis leaves include: Deionized water was added to the L-carnitine taurine ionic liquid to dilute it, resulting in an aqueous solution of L-carnitine taurine ionic liquid. The aqueous solution of L-carnitine taurine ionic liquid was then mixed with Platycladus orientalis leaves.

3. The method for preparing supramolecular Platycladus orientalis extract according to claim 2, characterized in that, The volume of the L-carnitine taurine ionic liquid accounts for 2% to 8% of the volume of the L-carnitine taurine ionic liquid aqueous solution.

4. The method for preparing supramolecular Platycladus orientalis extract according to claim 2, characterized in that, The ratio of the mass of the arborvitae leaves to the volume of the L-carnitine taurine ionic liquid aqueous solution is 1 g:(10~30) mL.

5. A supramolecular arborvitae extract, characterized in that, It was prepared using the method for preparing supramolecular arborvitae extract as described in any one of claims 1-4.

6. The application of the supramolecular arborvitae extract as described in claim 5 in the preparation of cosmetics.

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