Tetrandrine liposome having high drug-loading capacity, and preparation method therefor and use thereof

Abstract

A tetrandrine liposome having high drug-loading capacity, and a preparation method therefor and a use thereof. The liposome comprises the following components in parts by weight: 1.5-2.5 parts of tetrandrine, 0.6-1 part of a pH regulator, 3-5 parts of lecithin, 0.1-0.3 parts of cholesterol, 3-6 parts of a cosolvent, 0.5-1 part of an antioxidant, 6-10 parts of grease, 25-30 parts of water, and 35-55 parts of a polyol. The drug-loading capacity of the tetrandrine liposome prepared in the present invention can be as high as 1.5-2.5%, which is significantly higher than that of tetrandrine liposomes prepared by existing methods.

Description

A tetrandrine liposome with high drug loading, and its preparation method and application Technical Field

[0001] The present invention belongs to the technical field of cosmetics, and specifically relates to a tetrandrine liposome with a high drug loading, a preparation method thereof, and an application thereof, and in particular relates to a tetrandrine-containing liposome with a high drug loading, a preparation method thereof, and an application of the tetrandrine-containing nanoliposome in cosmetics. Background Art

[0002] Stephania tetrandra (Stephania tetrandra) is the dried root of Stephania tetrandra S. Moore, a plant of the Menispermaceae family. It dispels wind and relieves pain, promotes diuresis, and reduces swelling. It is used to treat rheumatic pain, edema, athlete's foot, dysuria, and eczema. Alkaloids are the main active ingredients in Stephania tetrandra, with tetrandrine and fangchinoline being the most abundant alkaloids. In cosmetics, tetrandrine's primary effects include anti-allergic and anti-inflammatory properties, analgesic and anti-swelling properties, and anti-redness and anti-itching. It is primarily used to treat various sensitive skin conditions.

[0003] Tetrandrine is poorly soluble in water and has low bioavailability, significantly impacting its use in cosmetics. Currently, commercially available cosmetic raw materials containing tetrandrine contain low levels. When added to cosmetics as an effective ingredient, tetrandrine levels typically range from approximately 10-100 ppm, limiting its effectiveness.

[0004] Liposomes are spherical, closed vesicles containing an aqueous phase, formed by phospholipids dispersed in water. Because liposomes have a phospholipid bilayer structure similar to that of cell membranes, they are also an important carrier for improving the solubility and bioavailability of tetrandrine. For example, Zhao Xiaodan [Zhao Xiaodan. Preparation of tetrandrine nanolipid carriers [J]. Chinese Journal of Health Care and Nutrition, 2012, (11): 4686-4687] prepared tetrandrine solid lipid nanoparticles (drug loading of 0.45%, encapsulation efficiency of 70%-90%) by emulsification-ultrasonic dispersion method; Hu Chunmei [Hu Chunmei, Zhu Li, Zhao Junyi, et al. Preparation and stability study of tetrandrine liposomes [J]. Chinese Pharmacy, 2008, 19(13): 995-997] prepared tetrandrine liposomes by ammonium sulfate gradient method (the second type of solvent chloroform was used in the process, the drug loading was 0.16%, and the encapsulation efficiency was 80.4%-81.6%); Chen Hongxuan [Chen Hongxuan, Wang Yanli. Determination of drug content and encapsulation efficiency of tetrandrine liposomes [J]. Journal of Henan University: Medical Edition, 2007, 26(4):38-40】The thin film dispersion-ultrasound method was used to prepare tetrandrine liposomes (the organic solvent anhydrous ethanol was used in the process, and the encapsulation efficiency was 69.2%-70.5%); Hu Jieru [Hu Jieru, Fu Junxia, ​​Zhu Yayan, et al. Experimental study on the prevention of diabetic cardiomyopathy by activating Nrf2 / HO-1 signaling pathway in novel tetrandrine nanoliposomes [J]. Chinese Pharmacist, 2021, 24(4):664-669] The thin film dispersion method combined with ultrasonic hydration technology was used to prepare tetrandrine nanoliposomes (the organic solvent anhydrous ethanol was used in the process, the drug loading was 0.1%, and the encapsulation efficiency was (87.23±2.21)%); Fan Chao [Fan Chao, Li Xinru, Zhou Yanxia, ​​et al. Preparation, characterization and anti-arthritis efficacy of tetrandrine nano-etholsomes [J]. Journal of Chinese Pharmaceutical Sciences, 2012, 21(4):311-320.】The pH gradient method was used to prepare tetrandrine ethosomes (drug loading of 0.15%, encapsulation efficiency of (52.87±3.81)%); Li Su [Li Su. Research on tetrandrine lipid nanoparticles [D]. Shenyang Pharmaceutical University, 2011] used the emulsification ultrasonic dispersion method to prepare tetrandrine solid lipid nanoparticles (drug loading of 0.54%, encapsulation efficiency of (89.01±1.61)%) and tetrandrine nanostructured lipid carriers (drug loading of 0.54%, encapsulation efficiency of (86.48±1.13)%). At present, there are three problems with tetrandrine liposomes:

[0005] (1) The preparation process uses toxic solvents such as chloroform, which is not suitable for application in the cosmetics field.

[0006] (2) The current liposome preparation method is more suitable for laboratory use rather than industrial-scale preparation for the following reasons: a. The thin film dispersion method is to dissolve the drug in an organic solvent, remove the solvent under reduced pressure, and form a thin film of lipids on the container wall. Then, a buffer solution containing a water-soluble drug is added and the liposome is obtained after sufficient shaking. However, this method requires the use of a large amount of organic solvents, is time-consuming, has a low encapsulation rate, and forms a large particle size. Moreover, the liposomes prepared industrially are affected by various factors such as negative pressure, temperature, and solvent volume, resulting in large batch differences. b. Ultrasonic dispersion method: Ultrasonic treatment is one of the earliest mechanical methods used to treat amphiphilic lipids: one is to insert the probe of the ultrasonic instrument into the liposome dispersion; the other is to place the sample in a test tube or beaker and then place it in a water bath ultrasonic instrument. This method is difficult to implement in industrial production, and because the ultrasonic intensity received at different positions of the equipment tank varies greatly, the liposome particle size obtained is very different and the process repeatability is poor. c. Chemical gradient method: The amphiphilic drug is lipid-soluble in the non-ionic state and can cross the lipid bilayer, but is hydrophilic in the ionized form and cannot cross the lipid bilayer to achieve drug encapsulation, mainly including pH. Gradient method, ammonium sulfate gradient method and calcium acetate gradient method can solve the problem of its industrial production to a certain extent; however, it requires the introduction of organic reagents and ionic reagents, which is prone to low encapsulation efficiency and high product ionic strength, especially affecting the preparation of cosmetic creams, emulsions and other dosage forms.

[0007] (3) The drug loading of tetrandrine in the current preparation process is relatively low, less than 0.6%.

[0008] Therefore, in view of the problems existing in the prior art, it is necessary to conduct research to provide a high-drug-loading liposome containing tetrandrine and a preparation method thereof, and apply the liposome containing tetrandrine to cosmetics to broaden the application field of tetrandrine liposome. Technical Solutions

[0009] In view of the deficiencies in the prior art, the present invention aims to provide a tetrandrine liposome with a high drug loading, a preparation method thereof and an application thereof.

[0010] The purpose of the present invention is achieved through the following technical solutions:

[0011] In a first aspect, the present invention provides a tetrandrine liposome with a high drug loading, comprising the following components in parts by weight:

[0012] 1.5-2.5 parts of tetrandrine,

[0013] pH regulator 0.6-1 part,

[0014] 3-5 parts of lecithin,

[0015] Cholesterol 0.1-0.3 parts,

[0016] 3 to 6 parts of cosolvent,

[0017] 0.5-1 part antioxidant,

[0018] 6-10 parts of oil,

[0019] 25-30 parts water,

[0020] 35-55 parts of polyol.

[0021] Preferably, the pH regulator is selected from at least one of lactic acid, citric acid, citric acid, tartaric acid and succinic acid.

[0022] More preferably, the pH adjuster is lactic acid.

[0023] Preferably, the mass ratio of tetrandrine to pH regulator is 1:0.4.

[0024] Preferably, the lecithin is soybean lecithin; the soybean lecithin is soybean lecithin CS-95.

[0025] Preferably, the cosolvent is selected from at least one of PEG-40 hydrogenated castor oil, PPG-13-decyltetradecyl alcohol polyether-24, and polyoxyethylene sorbitan monooleate.

[0026] More preferably, the cosolvent is PEG-40 hydrogenated castor oil.

[0027] Preferably, when the content of tetrandrine is 2-2.5%, the mass ratio of lecithin to cosolvent is 1:1.

[0028] Preferably, the antioxidant is selected from at least one of tocopherol, tocopherol acetate, ascorbyl stearate, ascorbyl tetraisopalmitate, tetrahexyldecyl ascorbate, and di-tert-butyl-p-cresol.

[0029] Preferably, the oil is selected from at least one of caprylic / capric triglyceride, glyceryl monostearate, glyceryl behenate, and glyceryl palmitostearate.

[0030] Preferably, the polyol is selected from at least one of glycerol, propylene glycol, butylene glycol, dipropylene glycol, and PEG400.

[0031] Preferably, the tetrandrine liposome with high drug loading further contains a preservative; the weight proportion of the preservative is 1 to 3.5 parts.

[0032] Preferably, the preservative is selected from at least one of 1,2-pentanediol, 1,2-hexanediol, p-hydroxyacetophenone, and phenoxyethanol.

[0033] In a second aspect, the present invention provides a method for preparing tetrandrine liposomes with a high drug loading, comprising the following steps:

[0034] A1. Stir lecithin, cosolvent, antioxidant, preservative, oil, cholesterol, and some polyols in a 70-75°C water bath until completely dissolved to obtain an oil phase;

[0035] A2. Sterilize water and add glycerol, stirring evenly; cool to below 50°C to obtain the aqueous phase;

[0036] A3. Add tetrandrine and pH adjuster to the oil phase, stir until completely dissolved, and then begin to cool to ≤50°C;

[0037] A4. Slowly add the mixture obtained after treatment in step A3 to the aqueous phase, stir and mix evenly to form a transparent liquid;

[0038] A5. Subject the transparent liquid to high-pressure homogenization to obtain tetrandrine liposomes.

[0039] In a second aspect, the present invention provides a use of the aforementioned high-drug-loading tetrandrine liposomes in the preparation of cosmetics.

[0040] In a fourth aspect, the present invention provides a use of the aforementioned high-drug-loading tetrandrine liposomes in the preparation of a soothing composition. Beneficial effects

[0041] Compared with the prior art, the present invention has the following beneficial effects:

[0042] 1. The drug loading capacity of the tetrandrine liposomes prepared by the present invention can be as high as 1.5-2.5%, which is significantly higher than that of the tetrandrine liposomes prepared by the existing method.

[0043] 2. The tetrandrine liposomes prepared by the present invention also have clear appearance, good stability, a stock solution particle size of 100-400 nm, a stock solution PDI ≤ 0.5 (preferably PDI ≤ 0.3), and an encapsulation efficiency ≥ 80% (preferably an encapsulation efficiency ≥ 90%), meeting the requirements for cosmetic use.

[0044] 3. After irritation test, hyaluronidase inhibition effect test, transdermal absorption test, and RAW264.7 cell inflammatory factor secretion inhibition test, the tetrandrine liposomes prepared by the present invention were proved to be fully applicable to cosmetics, which broadened the application field of tetrandrine liposomes. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Other features, objects and advantages of the present invention will become more apparent upon reading the detailed description of non-limiting embodiments with reference to the following drawings:

[0046] FIG1 shows the transdermal absorption effect of the tetrandrine liposomes prepared in Example 1; “*” indicates that compared with the tetrandrine extract solution (2.0%), *P<0.05;

[0047] Figure 2 shows the effect of the tetrandrine liposomes prepared in Example 1 on the activity of RAW264.7 cells; wherein, "###" indicates P < 0.001 compared with the Control group; "*" indicates P < 0.05 compared with the LPS control group; "**" indicates P < 0.01 compared with the LPS control group; and "***" indicates P < 0.001 compared with the LPS control group. Modes for Carrying Out the Invention

[0048] Type here a paragraph describing embodiments of the invention.

[0049] The present invention will be described in detail below with reference to specific embodiments. The following embodiments will help those skilled in the art to further understand the present invention, but are not intended to limit the present invention in any form. It should be noted that, for those skilled in the art, several variations and improvements can be made without departing from the scope of the present invention. These all fall within the scope of protection of the present invention.

[0050] The terms "preferred," "preferably," "more preferred," and the like, used herein, refer to embodiments of the invention that may provide certain benefits under certain circumstances. However, other embodiments may also be preferred under the same or other circumstances. Furthermore, the recitation of one or more preferred embodiments does not imply that other embodiments are not useful, nor is it intended to exclude other embodiments from the scope of the invention.

[0051] It should be understood that, except in any operating examples, or where otherwise indicated, all numbers indicating, for example, the amounts of ingredients used in the description and claims should be understood to be modified in all cases by the term "about". Therefore, unless otherwise indicated, the numerical parameters set forth in the following description and the appended claims are approximate values ​​that vary according to the desired properties to be obtained by the present invention. At least without attempting to limit the application of the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be interpreted in light of the number of reported significant figures and by applying ordinary rounding techniques.

[0052] Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values ​​set forth in the specific examples are reported as precisely as possible. Any numerical value, however, inherently contains certain errors necessarily resulting from the standard deviation found in their respective testing measurements.

[0053] In the following examples, all raw materials used are commercially available. The specific lecithin and preservatives used are described as follows:

[0054] Lecithin 1 was soybean lecithin CS-95, purchased from Avituo (Shanghai) Pharmaceutical Technology Co., Ltd.

[0055] Lecithin 2 is soybean lecithin purchased from Senrui Company;

[0056] Preservative 1 is a mixture of 1,2-pentanediol and 1,2-hexanediol, with a mixing ratio of 3:1;

[0057] Preservative 2 is a mixture of p-hydroxyacetophenone and 1,2-hexanediol, with the mixing ratio of the two being 1:1.

[0058] Example 1

[0059] This embodiment provides a tetrandrine liposome with a high drug loading capacity, the composition and weight proportions of the raw materials are shown in Table 1.

[0060] The preparation steps of the high drug loading tetrandrine liposomes are as follows:

[0061] 1) Stir lecithin 1, PPG-13-decyltetradecyl ether-24, tocopheryl acetate, preservative 1, caprylic / capric triglyceride, cholesterol, and propylene glycol in a 70-75°C water bath until completely dissolved to obtain an oil phase;

[0062] 2) Sterilize purified water at 80-90°C for 15 minutes, then add glycerol and stir evenly; cool to below 50°C to obtain the aqueous phase;

[0063] 3) Add tetrandrine and lactic acid to the oil phase and stir until completely dissolved. Stir for ≤10 minutes.

[0064] 4) Add the aqueous phase to the mixture treated in step 3 and stir for about 10 minutes to form a transparent liquid;

[0065] 5) The transparent liquid is subjected to high-pressure homogenization, specifically homogenization at 300 bar once, 500 bar once, and 800 bar three times, to obtain tetrandrine liposomes with a high drug loading.

[0066] Example 2

[0067] This embodiment provides a tetrandrine liposome with a high drug loading capacity, the composition and weight proportions of the raw materials are shown in Table 1.

[0068] The preparation steps of the high drug loading tetrandrine liposomes are as follows:

[0069] 1) Stir lecithin 1, PPG-13-decyltetradecyl ether-24, tocopheryl acetate, preservative 1, caprylic / capric triglyceride, cholesterol, and propylene glycol in a 70-75°C water bath until completely dissolved to obtain an oil phase;

[0070] 2) Sterilize purified water at 80-90°C for 15 minutes, then add glycerol and stir evenly; cool to below 50°C to obtain the aqueous phase;

[0071] 3) Add tetrandrine and lactic acid to the oil phase and stir until completely dissolved. Stir for ≤10 minutes.

[0072] 4) Add the aqueous phase to the mixture treated in step 3 and stir for about 10 minutes to form a transparent liquid;

[0073] 5) The transparent liquid is subjected to high-pressure homogenization, specifically homogenization at 300 bar once, 500 bar once, and 800 bar three times, to obtain tetrandrine liposomes with a high drug loading.

[0074] Example 3

[0075] This embodiment provides a tetrandrine liposome with a high drug loading capacity, the composition and weight proportions of the raw materials are shown in Table 1.

[0076] The preparation steps of the high drug loading tetrandrine liposomes are as follows:

[0077] 1) Stir lecithin 1, PPG-13-decyltetradecyl ether-24, tocopheryl acetate, preservative 1, caprylic / capric triglyceride, cholesterol, and propylene glycol in a 70-75°C water bath until completely dissolved to obtain an oil phase;

[0078] 2) Sterilize purified water at 80-90°C for 15 minutes, then add glycerol and stir evenly; cool to below 50°C to obtain the aqueous phase;

[0079] 3) Add tetrandrine and lactic acid to the oil phase and stir until completely dissolved. Stir for ≤10 minutes.

[0080] 4) Add the aqueous phase to the mixture treated in step 3 and stir for about 10 minutes to form a transparent liquid;

[0081] 5) The transparent liquid is subjected to high-pressure homogenization, specifically homogenization at 300 bar once, 500 bar once, and 800 bar three times, to obtain tetrandrine liposomes with a high drug loading.

[0082] Example 4

[0083] This embodiment provides a tetrandrine liposome with a high drug loading capacity, and the raw material components and weight proportions thereof are shown in Table 1.

[0084] The preparation steps of the high drug loading tetrandrine liposomes are as follows:

[0085] 1) Stir lecithin 1, PEG-40 hydrogenated castor oil, tocopheryl acetate, preservative 1, caprylic / capric triglyceride, cholesterol, and propylene glycol in a 70-75°C water bath until completely dissolved to obtain the oil phase;

[0086] 2) Sterilize purified water at 80-90°C for 15 minutes, then add glycerol and stir evenly; cool to below 50°C to obtain the aqueous phase;

[0087] 3) Add tetrandrine and lactic acid to the oil phase and stir until completely dissolved. Stir for ≤10 minutes.

[0088] 4) Add the aqueous phase to the mixture treated in step 3 and stir for about 10 minutes to form a transparent liquid;

[0089] 5) The transparent liquid is subjected to high-pressure homogenization, specifically homogenization at 300 bar once, 500 bar once, and 800 bar three times, to obtain tetrandrine liposomes with a high drug loading.

[0090] Example 5

[0091] This embodiment provides a tetrandrine liposome with a high drug loading capacity, and the raw material components and weight proportions thereof are shown in Table 1.

[0092] The preparation steps of the high drug loading tetrandrine liposomes are as follows:

[0093] 1) Stir lecithin 1, PEG-40 hydrogenated castor oil, tocopheryl acetate, preservative 1, caprylic / capric triglyceride, cholesterol, and propylene glycol in a 70-75°C water bath until completely dissolved to obtain the oil phase;

[0094] 2) Sterilize purified water at 80-90°C for 15 minutes, then add glycerol and stir evenly; cool to below 50°C to obtain the aqueous phase;

[0095] 3) Add tetrandrine and lactic acid to the oil phase and stir until completely dissolved. Stir for ≤10 minutes.

[0096] 4) Add the aqueous phase to the mixture treated in step 3 and stir for about 10 minutes to form a transparent liquid;

[0097] 5) The transparent liquid is subjected to high-pressure homogenization, specifically homogenization at 300 bar once, 500 bar once, and 800 bar three times, to obtain tetrandrine liposomes with a high drug loading.

[0098] Example 6

[0099] This embodiment provides a tetrandrine liposome with a high drug loading capacity, and the raw material components and weight proportions thereof are shown in Table 1.

[0100] The preparation steps of the high drug loading tetrandrine liposomes are as follows:

[0101] 1) Stir lecithin 1, PEG-40 hydrogenated castor oil, tocopheryl acetate, preservative 1, caprylic / capric triglyceride, cholesterol, and propylene glycol in a 70-75°C water bath until completely dissolved to obtain the oil phase;

[0102] 2) Sterilize purified water at 80-90°C for 15 minutes, then add glycerol and stir evenly; cool to below 50°C to obtain the aqueous phase;

[0103] 3) Add tetrandrine and lactic acid to the oil phase and stir until completely dissolved. Stir for ≤10 minutes.

[0104] 4) Add the aqueous phase to the mixture treated in step 3 and stir for about 10 minutes to form a transparent liquid;

[0105] 5) The transparent liquid is subjected to high-pressure homogenization, specifically homogenization at 300 bar once, 500 bar once, and 800 bar three times, to obtain tetrandrine liposomes with a high drug loading.

[0106] Example 7

[0107] This embodiment provides a tetrandrine liposome with a high drug loading capacity, and the raw material components and weight proportions thereof are shown in Table 1.

[0108] The preparation steps of the high drug loading tetrandrine liposomes are as follows:

[0109] 1) Stir lecithin 1, PEG-40 hydrogenated castor oil, tocopheryl acetate, preservative 1, caprylic / capric triglyceride, cholesterol, and propylene glycol in a 70-75°C water bath until completely dissolved to obtain the oil phase;

[0110] 2) Sterilize purified water at 80-90°C for 15 minutes, then add glycerol and stir evenly; cool to below 50°C to obtain the aqueous phase;

[0111] 3) Add tetrandrine and lactic acid to the oil phase and stir until completely dissolved. Stir for ≤10 minutes.

[0112] 4) Add the aqueous phase to the mixture treated in step 3 and stir for about 10 minutes to form a transparent liquid;

[0113] 5) The transparent liquid is subjected to high-pressure homogenization, specifically homogenization at 300 bar once, 500 bar once, and 800 bar three times, to obtain tetrandrine liposomes with a high drug loading.

[0114] Table 1

[0115]

[0116] Comparative Example 1

[0117] This comparative example provides a tetrandrine liposome with a high drug loading, the raw material components and mass percentages of which are shown in Table 2. Compared with Example 4, the only difference is that lecithin 1 is replaced by lecithin 2.

[0118] The preparation steps of the high drug loading tetrandrine liposomes are as follows:

[0119] 1) Stir lecithin 2, PEG-40 hydrogenated castor oil, tocopheryl acetate, preservative 1, caprylic / capric triglyceride, cholesterol, and propylene glycol in a 70-75°C water bath until completely dissolved to obtain the oil phase;

[0120] 2) Heat purified water to 80-90°C for sterilization for 15 minutes, then add glycerol and stir evenly; cool to below 50°C to obtain the aqueous phase;

[0121] 3) Add tetrandrine and lactic acid to the oil phase and stir until completely dissolved. Stir for ≤10 minutes.

[0122] 4) Add the aqueous phase to the mixture treated in step 3 and stir for about 10 minutes to form a transparent liquid;

[0123] 5) The transparent liquid is subjected to high-pressure homogenization, specifically homogenization at 300 bar once, 500 bar once, and 800 bar three times, to obtain tetrandrine liposomes with a high drug loading.

[0124] Comparative Example 2

[0125] This comparative example provides a tetrandrine liposome with a high drug loading, the raw material components and mass percentages of which are shown in Table 2. Compared with Example 4, the only difference is that lactic acid is not added.

[0126] The preparation steps of the high drug loading tetrandrine liposomes are as follows:

[0127] 1) Stir lecithin 2, PEG-40 hydrogenated castor oil, tocopheryl acetate, preservative 1, caprylic / capric triglyceride, cholesterol, and propylene glycol in a 70-75°C water bath until completely dissolved to obtain the oil phase;

[0128] 2) Heat purified water to 80-90°C for sterilization for 15 minutes, then add glycerol and stir evenly; cool to below 50°C to obtain the aqueous phase;

[0129] 3) Add tetrandrine to the oil phase and stir until completely dissolved. Stir for ≤10 minutes.

[0130] 4) Add the aqueous phase to the mixture treated in step 3 and stir for about 10 minutes to form a transparent liquid;

[0131] 5) The transparent liquid is subjected to high-pressure homogenization, specifically homogenization at 300 bar once, 500 bar once, and 800 bar three times, to obtain tetrandrine liposomes with a high drug loading.

[0132] Comparative Example 3

[0133] This comparative example provides a tetrandrine liposome with a high drug loading, the raw material components and mass percentages of which are shown in Table 2. Compared with Example 4, the only difference is that the mass percentage of lactic acid added is different.

[0134] The preparation steps of the high drug loading tetrandrine liposomes are as follows:

[0135] 1) Stir lecithin 2, PEG-40 hydrogenated castor oil, tocopheryl acetate, preservative 1, caprylic / capric triglyceride, cholesterol, and propylene glycol in a 70-75°C water bath until completely dissolved to obtain the oil phase;

[0136] 2) Heat purified water to 80-90°C for sterilization for 15 minutes, then add glycerol and stir evenly; cool to below 50°C to obtain the aqueous phase;

[0137] 3) Add tetrandrine and lactic acid to the oil phase and stir until completely dissolved. Stir for ≤10 minutes.

[0138] 4) Add the aqueous phase to the mixture treated in step 3 and stir for about 10 minutes to form a transparent liquid;

[0139] 5) The transparent liquid is subjected to high-pressure homogenization, specifically homogenization at 300 bar once, 500 bar once, and 800 bar three times, to obtain tetrandrine liposomes with a high drug loading.

[0140] Comparative Example 4

[0141] This comparative example provides a tetrandrine liposome with a high drug loading, the raw material components and mass percentages of which are shown in Table 2. Compared with Example 4, the only difference is that the mass percentage of lactic acid added is different.

[0142] The preparation steps of the high drug loading tetrandrine liposomes are as follows:

[0143] 1) Stir lecithin 2, PEG-40 hydrogenated castor oil, tocopheryl acetate, preservative 1, caprylic / capric triglyceride, cholesterol, and propylene glycol in a 70-75°C water bath until completely dissolved to obtain the oil phase;

[0144] 2) Heat purified water to 80-90°C for sterilization for 15 minutes, then add glycerol and stir evenly; cool to below 50°C to obtain the aqueous phase;

[0145] 3) Add tetrandrine and lactic acid to the oil phase and stir until completely dissolved. Stir for ≤10 minutes.

[0146] 4) Add the aqueous phase to the mixture treated in step 3 and stir for about 10 minutes to form a transparent liquid;

[0147] 5) The transparent liquid is subjected to high-pressure homogenization, specifically homogenization at 300 bar once, 500 bar once, and 800 bar three times, to obtain tetrandrine liposomes with a high drug loading.

[0148] Comparative Example 5

[0149] This comparative example provides a tetrandrine liposome with a high drug loading, the raw material components and mass percentages of which are shown in Table 2. Compared with Example 5, the only difference is that the mass percentage of PEG-40 hydrogenated castor oil added is different.

[0150] The preparation steps of the high drug loading tetrandrine liposomes are as follows:

[0151] 1) Stir lecithin 2, PEG-40 hydrogenated castor oil, tocopheryl acetate, preservative 1, caprylic / capric triglyceride, cholesterol, and propylene glycol in a 70-75°C water bath until completely dissolved to obtain the oil phase;

[0152] 2) Heat purified water to 80-90°C for sterilization for 15 minutes, then add glycerol and stir evenly; cool to below 50°C to obtain the aqueous phase;

[0153] 3) Add tetrandrine and lactic acid to the oil phase and stir until completely dissolved. Stir for ≤10 minutes.

[0154] 4) Add the aqueous phase to the mixture treated in step 3 and stir for about 10 minutes to form a transparent liquid;

[0155] 5) The transparent liquid is subjected to high-pressure homogenization, specifically homogenization at 300 bar once, 500 bar once, and 800 bar three times, to obtain tetrandrine liposomes with a high drug loading.

[0156] Comparative Example 6

[0157] This comparative example provides a tetrandrine liposome with a high drug loading, the raw material components and mass percentages of which are shown in Table 2. Compared with Example 5, the only difference is that the mass percentage of PEG-40 hydrogenated castor oil added is different.

[0158] The preparation steps of the high drug loading tetrandrine liposomes are as follows:

[0159] 1) Stir lecithin 2, PEG-40 hydrogenated castor oil, tocopheryl acetate, preservative 1, caprylic / capric triglyceride, cholesterol, and propylene glycol in a 70-75°C water bath until completely dissolved to obtain the oil phase;

[0160] 2) Heat purified water to 80-90°C for sterilization for 15 minutes, then add glycerol and stir evenly; cool to below 50°C to obtain the aqueous phase;

[0161] 3) Add tetrandrine and lactic acid to the oil phase and stir until completely dissolved. Stir for ≤10 minutes.

[0162] 4) Add the aqueous phase to the mixture treated in step 3 and stir for about 10 minutes to form a transparent liquid;

[0163] 5) The transparent liquid is subjected to high-pressure homogenization, specifically homogenization at 300 bar once, 500 bar once, and 800 bar three times, to obtain tetrandrine liposomes with a high drug loading.

[0164] Table 2

[0165]

[0166] Performance testing:

[0167] The tetrandrine liposomes prepared in Examples 1-7 and Comparative Examples 1-6 were tested for appearance, pH, particle size, PDI, zeta potential, stability, and encapsulation efficiency. The pH value was tested using a Raman pH meter, and the particle size and zeta potential were tested using a nanoparticle size and potential analyzer NS-90Z.

[0168] The specific method of the stability test is as follows: the example samples are placed in a constant temperature box and a light box at 4°C and 45°C for 3 months, and the properties of the samples are observed to see if they change. If no changes occur under the above conditions, the stability is recorded as "stable"; if changes occur, the state of the change is recorded.

[0169] The specific test method for encapsulation efficiency is:

[0170] (1) Total content determination method

[0171] Accurately measure 1.0 mL of tetrandrine liposomes and place it in a 20 mL volumetric flask. Add an appropriate amount of methanol and sonicate for 5 minutes to completely disrupt the ethosomes. Then dilute to volume with methanol, shake well, and filter through a 0.45 μm microporous membrane. The filtrate is used as the test solution. Accurately measure 20 μL of the test solution and inject it into a liquid chromatograph. Record the chromatogram and measure the area of ​​the main tetrandrine peak. Calculate the total content using the main peak area using the external standard method.

[0172] (2) Encapsulation efficiency determination method

[0173] Accurately measure 0.5 g of tetrandrine liposomes and place them in a centrifugal ultrafiltration tube. Place them in an ultracentrifuge at 4°C and 7000 r / min. -1 Centrifuge for 30 minutes. Transfer the filtrate to a 10 mL volumetric flask, dissolve it in methanol, dilute to the mark, and shake well. Filter through a 0.45 μm microporous filter membrane. Aspirate 20 μL and inject it into a liquid chromatograph. Record the chromatogram and measure the main peak area of ​​tetrandrine. Calculate the free drug content in the supernatant using the main peak area using the external standard method.

[0174] The encapsulation efficiency of tetrandrine liposomes was calculated according to the formula = (total content - free content in supernatant) / total content × 100%

[0175] In addition, the drug loading refers to the percentage of tetrandrine added when preparing tetrandrine liposomes. For example, if 2 parts by weight of tetrandrine are used in preparing 100 parts by weight of tetrandrine liposomes in Example 1, the drug loading in Example 1 is 2%.

[0176] The test results are shown in Table 3.

[0177] Table 3

[0178] Cosmetic application test:

[0179] 1) Tetrandrine liposomes prepared using Examples 1-3 were subjected to an irritation test using the chick chorioallantoic membrane (CAM) vascular test. The specific testing method was as follows: 10-11 day old chick embryos were selected. Using an egg candling device, the air cell location was marked. The eggshell layer above the air cell was peeled off with tweezers to expose the white shell membrane. An appropriate amount of physiological saline was added dropwise to moisten the shell membrane. The shell membrane was carefully removed with tweezers, ensuring that the vascular membrane was not damaged. A Teflon ring was placed in an area with dense blood vessels. 40 μl of the test sample was placed inside the ring and incubated in a constant temperature and humidity chamber for 30 minutes. Vascular damage within the Teflon ring was evaluated and recorded. Vascular responses in the chick chorioallantoic membrane (CAM) were scored. Four to ten valid chick embryos were used for each test sample. The irritation scores of the samples were recorded and a weighted average was calculated using the following formula. This average was the NC value (normal use concentration irritation value). The test results are shown in Table 4 below.

[0180] Table 4

[0181]

[0182] As shown in Table 4, the tetrandrine liposomes prepared in Examples 1-3 with drug loadings of 2%, 2.5%, and 1.5% respectively were diluted to 10% with PBS solution and subjected to chick embryo chorioallantoic membrane vascular experiments, which showed no vascular damaging stimulation.

[0183] 2) The tetrandrine liposomes prepared in Examples 1-3 were tested for their hyaluronidase inhibitory effect. The specific testing method was as follows:

[0184] a) Prior to the test, prepare 500 U / mL hyaluronidase, 0.5 mg / mL sodium hyaluronate, pH 5.6 sodium acetate buffer, acetylacetone solution, P-DAB color developer, calcium chloride, and sodium hydroxide solution in sequence according to the laboratory hyaluronidase test method.

[0185] b) The experiment was conducted in a 96-well plate and divided into a sample group (A), a sample zero adjustment group (B), a normal group (C), and a normal zero adjustment group (D). Initially, each group was treated with reagents (Group A: 25 μL sample + 25 μL hyaluronidase; Group B: 25 μL sample + 25 μL buffer; Group C: 25 μL distilled water + 25 μL hyaluronidase; Group D: 25 μL hyaluronidase + 25 μL buffer) and then incubated at 37°C for 20 minutes.

[0186] c) Add 5 μL of calcium chloride and continue to incubate at 37°C for 20 min.

[0187] d) Add 25 μL of sodium hyaluronate to the wells of Groups A and C, and add 25 μL of buffer to the wells of Groups B and D. Incubate at 37°C for 40 min.

[0188] e) Add 25 μL of distilled water, 5 μL of sodium hydroxide solution, and 25 μL of acetylacetone solution in that order. Place in a boiling water bath for 15 minutes, in an ice bath for 10 minutes, and then at room temperature for 10 minutes.

[0189] f) Add 50 μL of P-DAB solution, shake thoroughly, add 100 μL of anhydrous ethanol and let it stand at room temperature for 30 minutes. Measure the absorbance at a wavelength of 570 nm.

[0190] The test results of hyaluronidase inhibition rate (%) at different concentrations are shown in Table 5 below.

[0191] Table 5

[0192]

[0193] From the results in Table 5, it can be seen that the tetrandrine liposomes prepared in Examples 1-3 with a drug loading of 2%, 2.5% and 1.5% respectively can significantly inhibit the activity of hyaluronidase at different concentrations (diluted to different concentrations with PBS solution) and have a skin soothing effect.

[0194] 3) The tetrandrine liposomes prepared in Example 1 (tetrandrine loading: 2%) were tested for transdermal efficacy. The specific testing method was as follows:

[0195] The bionic skin used in the experiment is Milipore Strat-MTM Membrane, with a diameter of 25 mm and an area of ​​4.9 cm 2 .

[0196] A Strat-M™ membrane was fixed between the receiving and donor chambers. 1000 μL each of a 2.0% solution of Stephania tetrandra liposomes and a 2.0% solution of Stephania tetrandra extract were placed in the donor chamber. A 0.1 M PBS buffer was used as the receiving solution, and the mixture was stirred at 300 rpm at 32°C for diffusion. 1 mL of the receiving solution was collected at 0, 2, 4, 6, 8, and 24 hours. At 24 hours, the Strat-M™ membrane was chopped into small pieces, placed in a centrifuge tube, and soaked in 1 mL of PBS for 30 minutes. The supernatant was then collected by centrifugation.

[0197] The cumulative drug permeation at different times was calculated using HPLC analysis.

[0198] A tetrandrine solution (prepared by dissolving tetrandrine in 0.8% lactic acid and 97.2% purified water) was used as a control. The results of transdermal absorption are shown in FIG1 , which shows that the tetrandrine liposomes prepared in the present invention have a better transdermal effect than the tetrandrine solution.

[0199] 4) The tetrandrine liposomes prepared in Example 1 (tetrandrine loading of 2%) were tested for LPS (lipopolysaccharide)-induced secretion of the inflammatory factor TNF-α in RAW264.7 cells. The specific testing method was as follows:

[0200] a) Take cells in good exponential growth phase, digest them, seed them into 12-well plates, and culture for 24 hours.

[0201] b) The complete medium in the wells was aspirated and PBS was added for starvation treatment for 3 h. The blank control group was then added with complete medium, the model group was added with LPS and complete medium, and the sample group was added with LPS, complete medium, and diluted sample, and cultured for 48 h.

[0202] c) Aspirate the cell culture supernatant, centrifuge, and store in a -80°C freezer.

[0203] d) Set up separate wells for standards and samples. Add 100μL of the standard or sample to each well, shake gently, cover with a plate sticker, and incubate at 37°C for 2 hours. Set up 3-4 replicate wells for each sample. Discard the liquid, spin dry, and wash the plate five times. Add 100μL of biotin-conjugated antibody working solution to each well, cover with a new plate sticker, and incubate at 37°C for 1 hour. Discard the liquid in the wells, spin dry, and wash the plate five times, soaking for 1 minute each time, 200μL / well, and spin dry. Add 100μL of horseradish peroxidase-conjugated avidin working solution to each well, cover with a new plate sticker, and incubate at 37°C for 1 hour. Discard the liquid in the wells, spin dry, and wash the plate five times, soaking for 1 minute each time, 200μL / well, and spin dry. Add 100μL of substrate solution to each well in sequence and develop color at 37°C in the dark for 15-30 minutes. Add 100μL of stop solution to each well in sequence to terminate the reaction. Within 15 minutes after the reaction was terminated, the OD value of each well was measured in sequence at 450 nm using a microplate reader.

[0204] LPS was used as a control group (Control), and TNF-α secretion was tested at the same time. The test results are shown in Figure 2. As can be seen from Figure 2, the tetrandrine liposomes (2.0%) prepared by the present invention significantly reduced the TNF-α content at all test concentrations, indicating that the tetrandrine liposomes (2.0%) have the effect of inhibiting TNF-α secretion.

[0205] In conclusion, liposome tetrandrine (2.0%) has a soothing effect.

[0206] It can be seen from the above cosmetic application test results that the tetrandrine liposomes with high drug loading prepared by the present invention can be applied to cosmetics.

[0207] The present invention has many specific application paths, and the above is only a preferred embodiment of the present invention. It should be noted that the above embodiments are only used to illustrate the present invention and are not intended to limit the scope of protection of the present invention. For those skilled in the art, several improvements can be made without departing from the principles of the present invention, and these improvements should also be considered as the scope of protection of the present invention.

Claims

1. A tetrandrine liposome with high drug loading, characterized in that: The composition comprises the following components in parts by weight: 1.5-2.5 parts of tetrandrine, pH adjuster 0.6-1 part, 3-5 parts of lecithin, Cholesterol 0.1-0.3 parts, 3 to 6 parts of co-solvent, 0.5-1 part of antioxidant, 6-10 parts of oil, 25-30 parts water, 35-55 parts of polyol.

2. The tetrandrine liposome with high drug loading according to claim 1, characterized in that: The pH regulator is selected from at least one of lactic acid, citric acid, citric acid, tartaric acid and succinic acid.

3. The tetrandrine liposome with high drug loading according to claim 2, characterized in that: The pH adjuster is lactic acid.

4. The tetrandrine liposome with high drug loading according to claim 1, 2 or 3, characterized in that: The mass ratio of tetrandrine to the pH regulator is 1:0.

4.

5. The high drug loading powder of Tetrandrakirishima liposome according to claim 1, characterized in that: The lecithin is soybean lecithin; the soybean lecithin is soybean lecithin CS-95; The cosolvent is selected from at least one of PEG-40 hydrogenated castor oil, PPG-13-decyltetradecyl alcohol polyether-24, and polyoxyethylene sorbitan monooleate.

6. The tetrandrine liposome with high drug loading according to claim 1 or 5, characterized in that: When the content of tetrandrine is 2-2.5%, the mass ratio of lecithin to the cosolvent is 1:

1.

7. The tetrandrine liposome with high drug loading according to claim 1, characterized in that: The antioxidant is selected from at least one of tocopherol, tocopherol acetate, ascorbyl stearate, ascorbyl tetraisopalmitate, tetrahexyldecyl ascorbate, and di-tert-butyl-p-cresol; The oil is selected from at least one of caprylic / capric triglyceride, glyceryl monostearate, glyceryl behenate, and glyceryl palmitostearate; The polyol is selected from at least one of glycerol, propylene glycol, butylene glycol, dipropylene glycol, and PEG400.

8. The tetrandrine liposome with high drug loading according to claim 1, characterized in that: The tetrandrine liposome with high drug loading also contains a preservative; the weight proportion of the preservative is 1 to 3.5 parts; The preservative is selected from at least one of 1,2-pentanediol, 1,2-hexanediol, p-hydroxyacetophenone and phenoxyethanol.

9. A method for preparing the tetrandrine liposome with high drug loading according to any one of claims 1 to 8, characterized in that: The following steps are involved: A1. Stir lecithin, cosolvent, antioxidant, preservative, oil, cholesterol and some polyols in a water bath at 70-75°C until completely dissolved to obtain an oil phase; A2. Sterilize water and add glycerol, stir evenly; cool to below 50°C to obtain water phase; A3. Add tetrandrine and pH adjuster to the oil phase, stir to dissolve them completely, and then start to cool to ≤50°C; A4, slowly add the mixture obtained after the treatment in step A3 into the aqueous phase, stir and mix evenly to form a transparent liquid; A5. The transparent liquid is subjected to high pressure homogenization to obtain tetrandrine liposomes.

10. Use of the tetrandrine liposome with high drug loading according to any one of claims 1 to 8 in the preparation of cosmetics.

11. Use of the tetrandrine liposome with high drug loading according to any one of claims 1 to 8 in the preparation of a soothing composition.

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