Cardamonin nano-liposome capable of promoting transdermal absorption as well as preparation method and application of cardamonin nano-liposome

Abstract

The invention relates to the technical field of nano-liposomes, and discloses cardamonin nano-liposomes for promoting transdermal absorption and a preparation method and application thereof, the cardamonin nano-liposomes comprise an inner phase and an outer phase, the inner phase comprises the following raw materials in parts by weight: 5-10 parts of cardamonin, 2-10 parts of lecithin and 5-20 parts of grease, and the outer phase comprises the following raw materials in parts by weight: 5-10 parts of cardamonin, 2-10 parts of lecithin and 5-20 parts of grease; the stabilizer is carboxymethyl chitosan; an external phase: 0.1-5 parts of a surfactant, 0.1-3 parts of a stabilizer, 10-20 parts of short-chain alcohol and 44.9-80 parts of water; the mixture of the external phase and the internal phase is sequentially subjected to homogenization treatment and microjet homogenization treatment, and the cardamonin nano-liposome for promoting transdermal absorption is prepared. The cardamonin nano-liposome for promoting transdermal absorption provided by the invention is a flexible liposome, and can moisten skin and reduce moisture loss on the skin surface; the transdermal amount and retention amount of the carried components are obviously improved; the permeability of the cardamonin to the skin cell membrane is enhanced.

Description

Technical Field

[0001] The invention belongs to the technical field of nano liposomes, and in particular relates to cardamomum nano liposomes that promote transdermal absorption, and a preparation method and application thereof. Background Art

[0002] The Chinese medicinal herb cardamom is the dried nearly mature seeds of the plant cardamom of the genus Zingiberaceae. It has the effects of strengthening the spleen and warming the stomach, relieving depression, and removing dampness and cold. Cardamonin (CAR), with a chemical formula of 2',4'-dimethyl-6'-methoxychalcone, is a chalcone compound with multiple biological activities. In 1910, cardamonin was extracted from cardamom and was subsequently found to exist in many Chinese herbal medicines, such as galangal, wormwood, pecan, green leaf cedar, horseradish, Polygonum multiflorum, etc., and has a wide range of sources.

[0003] Studies have found that cardamomum can downregulate the expression of tyrosinase and inhibit the production of melanin, making it a potential whitening agent. The skin is a complex structure composed of the epidermis and dermis. External factors such as environmental pollution and ultraviolet rays can lead to an increase in skin free radicals and reactive harmful oxygen, and induce inflammatory damage, causing the skin to become thinner, wrinkles to increase, skin tone to darken, and skin to become sensitive. Cardamomum can remove free radicals in the skin, decompose hydrogen peroxide and peroxide, prevent the peroxidation of cell membrane lipids, reduce cell damage caused by oxidative stress, and regulate inflammatory signaling pathways, mediate the production of inflammatory factors, inhibit skin inflammatory reactions, inhibit photoaging at the cellular level, promote skin metabolism, reduce pigmentation, and protect the skin from damage by external factors.

[0004] However, the solubility of cardamomum is less than 2.7 μg / mL, which is almost insoluble in water, resulting in poor permeability on the surface of human and animal bodies, which greatly limits its effect and bioavailability. Summary of the invention

[0005] The present invention provides cardamomum nanoliposomes for promoting transdermal absorption and a preparation method and application thereof, which solve the problem in the prior art that cardamomum is almost insoluble in water, resulting in poor permeability on the surface of human and animal bodies, and the effect and bioavailability are highly limited.

[0006] The first object of the present invention is to protect a cardamom nanoliposome that promotes transdermal absorption, which consists of an inner phase and an outer phase, and the inner phase and the outer phase are composed of the following raw materials in parts by weight:

[0007] Internal phase: 5-10 parts of cardamomum, 2-10 parts of lecithin, 5-20 parts of oil;

[0008] External phase: 0.1-5 parts of surfactant, 0.1-3 parts of stabilizer, 10-20 parts of short-chain alcohol, 44.9-80 parts of water; the stabilizer is carboxymethyl chitosan;

[0009] The mixture of the external phase and the internal phase is sequentially subjected to homogenization treatment and microfluidization homogenization treatment to obtain the cardamomum nanoliposome.

[0010] Preferably, the internal phase comprises: 5-10 parts of cardamomum, 2-10 parts of lecithin, and 5-20 parts of oil;

[0011] External phase: 0.1-5 parts of surfactant, 0.1-3 parts of stabilizer, 10-20 parts of short-chain alcohol, and 50-80 parts of water.

[0012] Preferably, the particle size of the cardamomum nanoliposome is 40 to 50 nm.

[0013] Preferably, the lecithin is one of soybean lecithin with a phosphatidylcholine content of 75 parts or more, hydrogenated lecithin, dipalmitoyl phosphatidylcholine, and dipalmitoyl phosphatidylethanolamine, preferably soybean lecithin with a phosphatidylcholine content of 90% or more, so that flexible liposomes can be formed more effectively.

[0014] Preferably, the surfactant is one of sodium deoxycholate, sodium di(laurylamideglutamine)lysine, and polysorbate 80, preferably sodium di(laurylamideglutamine)lysine.

[0015] Preferably, the short-chain alcohol is one or more of propylene glycol, glycerol, butylene glycol, pentanediol or hexylene glycol. Short-chain alcohols can promote the fusion of lipid bilayers and improve the performance of liposomes.

[0016] Preferably, the oil is caprylic / capric triglyceride or squalane, so as to improve the performance of liposomes.

[0017] The second object of the present invention is to protect the preparation method of the cardamomum nanoliposome, comprising:

[0018] Heat the formulated amount of lecithin, cardamom and oil to 40-70°C, and stir magnetically for 15-25 minutes to mix evenly to form an internal phase;

[0019] Heat the formulated amount of surfactant, stabilizer, short-chain alcohol and water to 50-80° C., and stir magnetically for 10-30 minutes to mix evenly to obtain an external phase;

[0020] The external phase and the internal phase are mixed to obtain a mixture, the mixture is homogenized to obtain pre-liposomes, and the pre-liposomes are subjected to microfluidization homogenization to obtain cardamomum nanoliposomes.

[0021] Preferably, during the homogenization process, the speed of the homogenizer is 10000-15000 rpm and the time is 10 min-20 min.

[0022] Preferably, during the microfluidization homogenization treatment, the microfluidization pressure is 20,000 to 30,000 psi, and the cycle is 3 to 5 times.

[0023] The third object of the present invention is to protect the use of the cardamomum nanoliposomes that promote transdermal absorption in the preparation of cosmetics.

[0024] Compared with the prior art, the present invention has the following beneficial effects:

[0025] 1. The cardamomum nanoliposomes provided by the present invention for promoting transdermal absorption are flexible liposomes, which are similar to biological membrane structures. On the one hand, they can moisturize the skin and reduce water loss on the skin surface; on the other hand, the lipid membrane of the cardamomum nanoliposomes has a high degree of deformability, and can completely pass through the skin pores that are several times smaller than itself, enter the basal layer and the dermis, and significantly increase the transdermal amount and retention amount of the components carried; the cardamomum nanoliposomes can also enhance the permeability of cardamomum to the skin cell membrane, thereby increasing the concentration of active substances in the skin cells. Therefore, the present invention effectively solves the defect of low bioavailability of cardamomum, so that it can better play its effect.

[0026] 2. The present invention provides a method for preparing cardamom nanoliposomes that promote transdermal absorption. When preparing cardamom nanoliposomes, the advantages of the two are combined by coating the cardamom nanoliposomes with carboxymethyl chitosan, forming a carrier system with multiple excellent properties; the obtained cardamom nanoliposomes have a strong effect while increasing the application stability and achieving a higher degree of environmental response. The modification of the stabilizer carboxymethyl chitosan can also enhance the permeability and sustained release of cardamom nanoliposomes on the surface of humans and animals, making the release of the active substance cardamom more durable and controllable. At the same time, the use of the "homogeneous shear-microjet homogenization" high-energy emulsification combined technology has the characteristics of high-speed impact, shearing, and cavitation of the emulsion system in the shortest time, and can form a uniform, stable and ultra-fine fluid. The prepared liposome emulsion has the advantages of small particle size, uniform dispersibility, and commercial production, and also provides better stability, improved storage quality, improved absorption quality, saving expensive additives, changing viscosity, and reducing reaction time. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 is the IC50 value of the positive control VC;

[0028] Figure 2 The ABTS free radical scavenging rate of cardamomum nanoliposomes prepared in different groups;

[0029] Figure 3 Cumulative transdermal permeability of cardamomum nanoliposomes prepared in different groups;

[0030] Figure 4 This is the cumulative release rate of cardamomum nanoliposomes prepared in different groups. DETAILED DESCRIPTION

[0031] The technical solutions in the present invention will be described clearly and completely below in conjunction with the specific implementation of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0032] The methods described in the embodiments of the present invention are conventional methods unless otherwise specified. The materials, reagents, etc. used can be obtained from commercial sources unless otherwise specified.

[0033] In the following description of this embodiment, the term "and / or" is used to describe the association relationship of associated objects, indicating that there may be three relationships. For example, A and / or B can represent: A exists alone, B exists alone, and A and B exist at the same time. Among them, A and B can be singular or plural; the single symbol " / " means "or".

[0034] In the following description of this embodiment, the term "at least one" refers to one or more, and "plurality" refers to two or more. "At least one of the following" or similar expressions refer to any combination of these items, including any combination of single items or plural items. For example, "at least one of A, B or C", or "at least one of A, B and C" can represent any one of A, B, C, or A+B, or A+C, or B+C, or A+B+C, where A, B, C can be single or multiple, respectively.

[0035] In the following description of this embodiment, the order of serial numbers does not mean the order of execution. Some or all of the steps can be executed in parallel or sequentially. The execution order of each process should be determined by its function and internal logic, and should not constitute any limitation on the implementation process of this embodiment.

[0036] In the following description of the present embodiment, the numerical range should be understood to also specifically disclose each intermediate value between the upper and lower limits of the range. Any stated value or intermediate value in the stated range and any other stated value or each smaller range between the intermediate values ​​in the stated range are also included in the present embodiment, and the upper and lower limits of the smaller range can be independently included or excluded in the range.

[0037] Unless otherwise stated, the technical / scientific terms used in the present embodiment have the same meanings as those generally understood by those skilled in the art to which the present application belongs. Although the present application only describes preferred methods and materials, any similar or equivalent methods and materials may also be used in the implementation or testing of the present application. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the documents. In the event of any conflict with any incorporated document, the content of this specification shall prevail.

[0038] Example 1

[0039] A cardamomum nanoliposome for promoting transdermal absorption, which consists of an inner phase and an outer phase, wherein the inner phase and the outer phase are composed of the following raw materials in parts by weight:

[0040] Internal phase: 5 parts of cardamomum, 6 parts of soy lecithin, 10 parts of caprylic / capric triglyceride;

[0041] External phase: 2.5 parts of sodium di(laurylamideglutamine)lysine, 2.5 parts of carboxymethyl chitosan, 15 parts of glycerol, and 59 parts of water.

[0042] The preparation method of the cardamomum nanoliposome comprises:

[0043] (1) 6 parts of soybean lecithin, 5 parts of cardamomum, and 10 parts of caprylic / capric triglyceride were mixed, heated to 50° C., and magnetically stirred for 20 minutes to form an internal phase.

[0044] (2) 2.5 parts of sodium di(laurylamide glutamine) lysine, 2.5 parts of carboxymethyl chitosan, 15 parts of glycerol and 59 parts of water were mixed, heated to 50° C., and magnetically stirred for 30 minutes to obtain an external phase after stirring.

[0045] (3) After mixing the inner phase and the outer phase, the mixture is homogenized, and the speed of the homogenizer is controlled to be 15000 rpm for 20 min to obtain pre-liposomes.

[0046] (4) The pre-liposomes were passed into a microfluidizer for microfluidization homogenization, the microfluidization pressure was controlled at 25000 psi, and the number of cycles was 5. Finally, after being processed by a series of liposome extruders, cardamomum nano-flexible liposomes with a particle size of 41.94 nm were obtained.

[0047] Example 2

[0048] A cardamomum nanoliposome for promoting transdermal absorption, which consists of an inner phase and an outer phase, wherein the inner phase and the outer phase are composed of the following raw materials in parts by weight:

[0049] Internal phase: 5 parts of cardamomum, 2 parts of soy lecithin, 5 parts of caprylic / capric triglyceride;

[0050] External phase: 0.1 parts of sodium di(laurylamide glutamine) lysine, 0.1 parts of carboxymethyl chitosan, 10 parts of short-chain alcohol, and 79.8 parts of water. The short-chain alcohol includes 7 parts of propylene glycol and 3 parts of glycerol.

[0051] The preparation method of the cardamomum nanoliposome comprises:

[0052] (1) 2 parts of soybean lecithin, 5 parts of cardamomum, and 5 parts of caprylic / capric triglyceride were mixed, heated to 40°C, and magnetically stirred for 15 minutes to form an internal phase.

[0053] (2) 0.1 parts of sodium di(laurylamide glutamine) lysine, 0.1 parts of carboxymethyl chitosan, 7 parts of propylene glycol, 3 parts of glycerol and 79.8 parts of water were mixed, heated to 50° C., and magnetically stirred for 10 minutes to obtain an external phase after stirring.

[0054] (3) After mixing the inner phase and the outer phase, the mixture is homogenized, and the speed of the homogenizer is controlled to be 10000 rpm for 10 min to obtain pre-liposomes.

[0055] (4) The pre-liposomes were passed into a microfluidizer for microfluidization homogenization, the microfluidization pressure was controlled at 20,000 psi, and the number of cycles was 3. Finally, after being processed by a series of liposome extruders, cardamomum nano-flexible liposomes with a particle size of 46.89 nm were obtained.

[0056] Example 3

[0057] A cardamomum nanoliposome for promoting transdermal absorption, which consists of an inner phase and an outer phase, wherein the inner phase and the outer phase are composed of the following raw materials in parts by weight:

[0058] Internal phase: 7 parts of cardamomum, 8 parts of hydrogenated lecithin, 15 parts of squalane;

[0059] External phase: 5 parts of sodium deoxycholate, 0.1 parts of carboxymethyl chitosan, 20 parts of butanediol, and 44.9 parts of water.

[0060] The preparation method of the cardamomum nanoliposome comprises:

[0061] (1) 8 parts of hydrogenated lecithin, 7 parts of cardamom and 15 parts of squalane were mixed, heated to 60° C., and magnetically stirred for 25 minutes to form an internal phase.

[0062] (2) 5 parts of sodium deoxycholate, 0.1 parts of carboxymethyl chitosan, 20 parts of butanediol and 44.9 parts of water were mixed, heated to 80° C., and magnetically stirred for 10 minutes to obtain an external phase after stirring.

[0063] (3) After mixing the inner phase and the outer phase, the mixture is homogenized, and the speed of the homogenizer is controlled to be 10000 rpm for 20 min to obtain pre-liposomes.

[0064] (4) The pre-liposomes were passed into a microfluidizer for microfluidization homogenization, the microfluidization pressure was controlled at 25000 psi, and the number of cycles was 3. Finally, after being processed by a liposome extruder connected in series, cardamomum nano-flexible liposomes with a particle size of 42.12 nm were obtained.

[0065] Example 4

[0066] A cardamomum nanoliposome for promoting transdermal absorption, which consists of an inner phase and an outer phase, wherein the inner phase and the outer phase are composed of the following raw materials in parts by weight:

[0067] Internal phase: 10 parts of cardamomum, 10 parts of dipalmitoylphosphatidylcholine, 20 parts of squalane;

[0068] External phase: 1 parts of polysorbate 80, 3 parts of carboxymethyl chitosan, 10 parts of pentanediol, and 46 parts of water.

[0069] The preparation method of the cardamomum nanoliposome comprises:

[0070] (1) 10 parts of dipalmitoylphosphatidylcholine, 10 parts of cardamomum, and 20 parts of squalane were mixed, heated to 70° C., and magnetically stirred for 25 minutes to form an internal phase.

[0071] (2) 1 part of polysorbate 80, 3 parts of carboxymethyl chitosan, 10 parts of pentanediol and pentanediol water were mixed, heated to 60° C., and magnetically stirred for 25 minutes to obtain an external phase after stirring.

[0072] (3) After mixing the inner phase and the outer phase, the mixture is homogenized, and the speed of the homogenizer is controlled to be 14000 rpm for 10 min to obtain pre-liposomes.

[0073] (4) The pre-liposomes were passed into a microfluidizer for microfluidization homogenization, the microfluidization pressure was controlled at 20,000 psi, and the number of cycles was 5. Finally, after being processed by a series of liposome extruders, cardamomite nano-flexible liposomes with a particle size of 44.07 nm were obtained.

[0074] Based on Example 1, a comparative example is set for comparison.

[0075] Comparative Example 1 (without adding stabilizer carboxymethyl chitosan)

[0076] A cardamomum nanoliposome, which consists of an inner phase and an outer phase, wherein the inner phase and the outer phase are composed of the following raw materials in parts by weight:

[0077] Internal phase: 5 parts of cardamomum, 6 parts of soy lecithin, 10 parts of caprylic / capric triglyceride;

[0078] External phase: 2.5 parts of sodium di(laurylamide glutamine) lysine, 15 parts of glycerol, and 59 parts of water.

[0079] The preparation method of the cardamomum nanoliposome comprises:

[0080] (1) 6 parts of soybean lecithin, 5 parts of cardamomum, and 10 parts of caprylic / capric triglyceride were mixed, heated to 50° C., and magnetically stirred for 20 minutes to form an internal phase.

[0081] (2) 2.5 parts of sodium di(laurylamide glutamine) lysine, 15 parts of glycerol and 59 parts of water were mixed, heated to 50° C., and magnetically stirred for 30 minutes to obtain an external phase after stirring.

[0082] (3) After mixing the inner phase and the outer phase, the mixture is homogenized, and the speed of the homogenizer is controlled to be 15000 rpm for 20 min to obtain pre-liposomes.

[0083] (4) The pre-liposomes were passed into a microfluidizer for microfluidization homogenization, the microfluidization pressure was controlled at 25000 psi, and the number of cycles was 5. Finally, after being processed by a series of liposome extruders, cardamomum nano-flexible liposomes with a particle size of 56.58 nm were obtained.

[0084] Comparative Example 2 (High-pressure homogenization)

[0085] A cardamomum nanoliposome, which consists of an inner phase and an outer phase, wherein the inner phase and the outer phase are composed of the following raw materials in parts by weight:

[0086] Internal phase: 5 parts of cardamomum, 6 parts of soy lecithin, 10 parts of caprylic / capric triglyceride;

[0087] External phase: 2.5 parts of sodium di(laurylamideglutamine)lysine, 2.5 parts of carboxymethyl chitosan, 15 parts of short-chain alcohol, and 59 parts of water.

[0088] The preparation method of the cardamomum nanoliposome comprises:

[0089] (1) 6 parts of soybean lecithin, 5 parts of cardamomum, and 10 parts of caprylic / capric triglyceride were mixed, heated to 50° C., and magnetically stirred for 20 minutes to form an internal phase.

[0090] (2) 2.5 parts of sodium di(laurylamide glutamine) lysine, 2.5 parts of carboxymethyl chitosan, 15 parts of glycerol and 59 parts of water were mixed, heated to 50° C., and magnetically stirred for 30 minutes to obtain an external phase after stirring.

[0091] (3) After the inner phase and the outer phase are mixed, they are treated by a high-pressure homogenizer, the pressure is controlled to be 2000 bar, the number of cycles is 5, and cardamomum nanoliposomes with a particle size of 80.25 nm are obtained.

[0092] Comparative Example 3 (without surfactant)

[0093] A cardamomum nanoliposome, which consists of an inner phase and an outer phase, wherein the inner phase and the outer phase are composed of the following raw materials in parts by weight:

[0094] Internal phase: 5 parts of cardamomum, 6 parts of soy lecithin, 10 parts of caprylic / capric triglyceride;

[0095] External phase: 2.5 parts of carboxymethyl chitosan, 15 parts of glycerol, and 59 parts of water.

[0096] The preparation method of the cardamomum nanoliposome comprises:

[0097] (1) 6 parts of soybean lecithin, 5 parts of cardamomum, and 10 parts of caprylic / capric triglyceride were mixed, heated to 50° C., and magnetically stirred for 20 minutes to form an internal phase.

[0098] (2) 2.5 parts of carboxymethyl chitosan, 15 parts of glycerol and 59 parts of water were mixed, heated to 50° C., and magnetically stirred for 20 minutes to obtain an external phase after stirring.

[0099] (3) After mixing the inner phase and the outer phase, the mixture is homogenized, and the speed of the homogenizer is controlled to be 15000 rpm for 20 min to obtain pre-liposomes.

[0100] (4) The pre-liposomes were passed into a microfluidizer for microfluidization homogenization, the microfluidization pressure was controlled at 25000 psi, and the number of cycles was 5. Finally, after being processed by a liposome extruder in series, cardamomum nano-flexible liposomes with a particle size of 59.42 nm were obtained.

[0101] Comparative Example 4 (liposomes not prepared)

[0102] Add 5 parts of cardamomum to 25 parts of glycerol, disperse it by ultrasonication at room temperature for 50 minutes, then add ultrapure water, stir and mix to obtain a free cardamomum solution.

[0103] Results

[0104] Experimental Example 1: Liposome Characterization Analysis

[0105] The cardamomum nanoliposomes provided in Examples 1 to 4 and Comparative Examples 1 to 3 were diluted 4 times with deionized water and placed in a 90plus PALS laser particle size analyzer to measure the average particle size, polydispersity coefficient and Zeta potential of the liposomes. The specific test results are shown in Table 1.

[0106] Table 1 Sample particle size, PDI and Zeta potential test

[0107]

[0108]

[0109] Note: “-” means no result.

[0110] The small particle size of the liposome increases the specific surface area and the bioavailability; the smaller the PDI, the more uniform the molecular weight distribution; the higher the absolute value of the Zeta potential, the more stable the system. As shown in Table 1, compared with Comparative Examples 1 to 3, the cardamomite nano-flexible liposomes prepared in Examples 1 to 4 of the present invention have a smaller average particle size, a smaller PDI, and a higher absolute value of the Zeta potential. Considering the stability of the cardamomite nano-liposomes during storage and the efficacy and activity during application, the cardamomite nano-flexible liposomes provided in the examples are more suitable.

[0111] Test Example 2: Stability Test

[0112] Since the stability of the cardamomite nanoliposomes prepared in Example 2 and Example 4 is basically the same as that of the cardamomite nanoliposomes prepared in Example 1 and Example 3, the following test is only performed using the cardamomite nanoliposomes prepared in Example 1 and Example 3 as an example.

[0113] The cardamomum nanoliposomes prepared in Example 1, Example 3, Comparative Example 1, Comparative Example 2, and Comparative Example 3 were respectively packaged in vials and placed under five conditions: -18°C, 4°C, room temperature, 40°C, and light for 30 days, and the appearance (whether there is agglomeration), encapsulation efficiency, and particle size of the liposomes were examined respectively. The samples placed at -18°C were taken out and thawed at room temperature before being examined again.

[0114] The cardamomum nanoliposomes prepared in Example 1, Example 3, Comparative Example 1, Comparative Example 2 and Comparative Example 3 were diluted 4 times with deionized water and placed in a 90plus PALS laser particle size analyzer to measure the average particle size of the liposomes.

[0115] The cardamomum nanoliposomes prepared in Example 1, Example 3, Comparative Example 1, Comparative Example 2, and Comparative Example 3 were respectively passed through a 0.22 μm organic filter membrane, and 100 μL was taken out for demulsification using methanol. The effective ingredient content (w1) of the cardamomum nanoliposomes and the mass of the active ingredient of dragon's blood A added when preparing the liposomes (w0) were measured by HPLC, and the encapsulation efficiency was calculated according to the formula: encapsulation efficiency = (w1 / w0) × 100%.

[0116] The specific test results are shown in Table 2.

[0117] Table 2 Results of the four conditions after 30 days of storage

[0118]

[0119]

[0120] Note: “-” means no result.

[0121] It can be seen from the results in Table 2 that compared with Comparative Example 1, Comparative Example 2, and Comparative Example 3, the cardamomite nanoliposomes prepared in Example 1 and Example 3 are stored under different temperature and light conditions, and the encapsulation rate and particle size change are smaller and the stability is higher. The cardamomite nanoliposomes prepared in Comparative Example 1 were placed at 40°C for 30 days, and a small amount of agglomeration occurred, while Examples 1 and 3 did not agglomerate under the same conditions, which shows that the modification of the stabilizer carboxymethyl chitosan increases the application stability and achieves a higher degree of environmental response. Considering the convenience of storage and use of cardamomite nanoliposomes, the cardamomite nano flexible liposomes provided in the embodiment are more suitable.

[0122] Test Example 3: Free Radical Scavenging Experiment

[0123] 10 μL of cardamomite nanoliposomes of Examples 1 to 4 and Comparative Examples 2 to 4 were mixed with 190 μL of 0.25mmol / L PBS solution, and shaken well to obtain the ABTS working solution of the sample. 10 μL of cardamomite nanoliposomes of Examples 1 to 4 and Comparative Examples 2 to 4 were mixed with 190 μL of 0.25mmol / L PBS solution, and shaken well to obtain the PBS solution of the sample. Incubate at room temperature for 6 minutes, and measure the absorbance at 405nm. The absorbance of the ABTS working solution of the sample is recorded as A1, the absorbance of the PBS solution of the sample is recorded as A2, and the absorbance of the blank ABTS working solution and the blank PBS is recorded as A3. The ABTS free radical scavenging rate is calculated according to the formula ABTS scavenging rate = [1-(A1-A2) / A3]×100%. A positive control group was set up, in which vitamin C was loaded at concentrations of 800 μg / mL, 400 μg / mL, 200 μg / mL, 100 μg / mL, 50 μg / mL, 25 μg / mL, 12.5 μg / mL, and 6.25 μg / mL. The results are shown in Table 3 and Figure 1 , 2 Results shown.

[0124] Table 3 ABTS free radical scavenging rate

[0125]

[0126]

[0127] When the IC50 of the positive control group is between 0.040 and 0.070 g / L, the system is considered effective. Figure 1 is the IC50 value of the positive control VC, Figure 1 It can be obtained that the IC50 of the positive control VC is 62.75 μg / mL, which is in the range of 0.040 to 0.070 g / L, so this test system is considered to be effective. Figure 2 The ABTS free radical scavenging rate of cardamomum nanoliposomes prepared in different groups is shown in Table 3. Figure 2 The results show that the ABTS free radical scavenging rate of Example 1 is the highest, with an average of 64.07%. The ABTS free radical scavenging rates of Examples 2, 3, and 4 are slightly lower but all higher than the comparative example. The free radical scavenging rate of Comparative Example 4 is an average of 13.19%. It can be seen that the application of cardamom is limited by its solubility problem. The cardamom nanoliposomes provided by the embodiments of the present invention can significantly improve the antioxidant properties of cardamom, showing obvious superiority.

[0128] Test Example 4: In vitro skin penetration test

[0129] Since the cumulative permeability of the cardamomite nanoliposomes prepared in Examples 2 to 4 is substantially the same as that of the cardamomite nanoliposomes prepared in Example 1, only Example 1 is taken as an example for comparison with the comparative example.

[0130] Place the Franz diffusion cell in a drug transdermal diffusion tester, and set the condition to a 37°C constant temperature circulating water bath. Spread the mouse skin flat between the supply cell and the receiving cell, and fix it with a stainless steel clip, with the skin stratum corneum facing the supply cell and the subcutaneous tissue facing the receiving cell. Fill the receiving cell with 6mL of receiving solution (PBS with pH = 7.2-7.4), exhaust the air bubbles, and equilibrate at 37°C and 300r / min for 1h. Add 1mL of the cardamom nanoliposomes of Example 1, Comparative Examples 1-3, and the cardamom solution sample prepared in Comparative Example 4 to the supply cell, respectively, and allow the complete components of the stratum corneum to contact the sample. Seal the supply cell with a sealing film to prevent the sample from volatilizing, and stir the receiving solution continuously to ensure that the sample is evenly dispersed; take out 1mL of the receiving solution at the set time points, and immediately add an equal amount of fresh receiving solution PBS to replenish it after each sampling. The samples taken out were subjected to HPLC determination and the cumulative permeation amount and cumulative permeability per unit skin area were calculated. The specific test results are shown as follows. Figure 3 shown.

[0131] Cumulative permeation (Q): = (A x V) / A; V is the volume of the receiving chamber (ml); A is the exposed area of ​​the skin sample (cm2).

[0132] Cumulative permeability (J): J = Q / (A×t), where Q is the amount of drug that permeates the skin in time t; A is the exposed area of ​​the skin sample (square centimeters); and t is the experimental time (hours).

[0133] Depend on Figure 3 The results show that after 24 hours of permeation experiment, the cumulative permeation amount and permeability of all samples through the mouse skin increased with time. After 24 hours, the cumulative transdermal permeability of Example 1 reached 5.3%, showing a better skin permeation effect than the comparative example. The cumulative transdermal permeability of Comparative Example 1 is small, proving that the modification of carboxymethyl chitosan in Example 1 can increase the permeability of liposomes; the reason for the decrease in the cumulative transdermal permeability of Comparative Example 2 may be that the particle size is large, which affects its ability to penetrate the skin; Comparative Example 3 is an ordinary nanoliposome, and its cumulative transdermal permeability is lower than that of Example 1, which may be due to the fact that the cardamom nanoliposomes prepared in Example 1 are flexible liposomes with high deformation ability, which can quickly pass through the skin pores and significantly increase the transdermal amount of the components carried; Comparative Example 4 is free cardamom, and the skin permeability is poor. It can be seen that the bioavailability of cardamom is significantly improved through the encapsulation of nanoliposomes.

[0134] Test Example 5: In vitro release test

[0135] 2 mL of the cardamom nano-flexible liposomes of Example 1 and the cardamom solution of Comparative Example 4 were loaded into a dialysis bag (Mw=14000 kDa). A release medium was prepared from Tween 80, ethanol and a phosphate buffer solution with a pH of 5.8; wherein the volume ratio of Tween 80, ethanol and phosphate buffer solution was 5:20:75, and the dialysis bag was placed in a 250 mL beaker filled with 200 mL of the release medium. The release medium was maintained at 32°C and stirred continuously at 200 rpm. At time points of 0, 1, 2, 4, 6, 8, 10, 12, and 24 hours, 3 mL of the release medium was taken and the same volume of fresh release medium was immediately added. The cumulative release of cardamom was measured over a certain period of time and plotted as a function of time. The specific test results are as follows Figure 4 shown.

[0136] Depend on Figure 4 The results show that the release rate of cardamomamine that has not been encapsulated (Comparative Example 4) reaches 85% at 24 hours. The release curve of cardamomamine in the liposomes encapsulated by nano-flexible liposomes (Example 1) is flat, and the release rate at 24 hours is 32.12%, indicating that the encapsulation of nano-flexible liposomes can significantly prolong the release time of cardamomamine and effectively increase the retention time in the body.

[0137] Test Example 6: Safety Test-Skin Patch Test

[0138] The cardamomite nano flexible liposome sample of Example 1 was used as the test group, and the free cardamomite solution sample of Comparative Example 4 was used as the control group. 0.03g of the sample was measured for safety and efficacy testing: 30 volunteers were selected, and the product was subjected to the "Human Skin Patch Test" in accordance with the "Technical Specifications for Safety of Cosmetics (2015 Edition)". The skin reaction grading standard of the skin occlusion patch test in Table 5 was used for rating at 0.5h and 24h, and the results are shown in Table 4.

[0139] Table 4 Skin occlusive patch test skin reaction grading standard

[0140]

[0141] Table 5 Statistics of skin patch test results

[0142]

[0143] From the statistical results in Table 5, it can be seen that the experimental group had a reaction rating of 0 at 0.5h and 24h, and no adverse skin reactions were caused, indicating that the cardamomite nano-flexible liposomes were highly safe. In Comparative Example 4, one subject showed a grade 1 reaction at 24h, which may be due to the uneven dispersion of the free cardamomite solution, resulting in excessive local concentration, which showed a slight irritation.

[0144] Although the preferred embodiments of the present invention have been described, those skilled in the art may make other changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

[0145] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. A cardamomum nanoliposome for promoting transdermal absorption, characterized in that: It consists of an inner phase and an outer phase, and the inner phase and the outer phase are composed of the following raw materials in parts by weight: Internal phase: 5-10 parts of cardamomum, 2-10 parts of lecithin, 5-20 parts of oil; External phase: 0.1-5 parts of surfactant, 0.1-3 parts of stabilizer, 10-20 parts of short-chain alcohol, 44.9-80 parts of water; the stabilizer is carboxymethyl chitosan; The mixture of the external phase and the internal phase is sequentially subjected to homogenization treatment and microfluidization homogenization treatment to obtain the cardamomum nanoliposome.

2. The cardamomum nanoliposome according to claim 1, characterized in that: The particle size of the cardamomum nanoliposome is 40 to 50 nm.

3. The cardamomum nanoliposome according to claim 1, characterized in that: The lecithin is one of soybean lecithin, hydrogenated lecithin, dipalmitoylphosphatidylcholine and dipalmitoylphosphatidylethanolamine.

4. The cardamomum nanoliposome according to claim 1, characterized in that: The surfactant is one of sodium deoxycholate, sodium di(laurylamide glutamine) lysine and polysorbate 80.

5. The cardamomum nanoliposome according to claim 1, characterized in that: The short-chain alcohol is one or more of propylene glycol, glycerol, butylene glycol, pentanediol or hexylene glycol.

6. The cardamomum nanoliposome according to claim 1, characterized in that: The oil is caprylic / capric triglyceride or squalane.

7. A method for preparing cardamomum nanoliposomes according to any one of claims 1 to 6, characterized in that: include: Mix the formulated amount of lecithin, cardamom and oil evenly to form an internal phase; The surfactant, stabilizer, short-chain alcohol and water in the formula are uniformly mixed to obtain an external phase; The external phase and the internal phase are mixed to obtain a mixture, and the mixture is subjected to homogenization treatment and microfluidization homogenization treatment in sequence to obtain cardamomum nanoliposomes.

8. The method for preparing cardamomum nanoliposomes according to claim 7, characterized in that: During the homogenization process, the speed of the homogenizer is 10000-15000 rpm, and the time is 10-20 minutes.

9. The method for preparing cardamomum nanoliposomes according to claim 7, characterized in that: During the microfluidic homogenization treatment, the microfluidic pressure is 20,000 to 30,000 psi, and the cycle is 3 to 5 times.

10. Use of the cardamomum nanoliposomes for promoting transdermal absorption according to claim 1 in the preparation of cosmetics.

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