A compound traditional Chinese medicine liposome gel patch for treating early-onset ovarian insufficiency and its preparation method.

Abstract

This invention discloses a compound traditional Chinese medicine liposome gel patch for treating early-onset ovarian insufficiency and its preparation method. The preparation method includes: 1) extracting the effective active ingredients from the compound traditional Chinese medicine and preparing them into solid particles; 2) loading the active ingredient particles into rationally designed nano-bilayer composite liposomes; 3) immobilizing the liposomes with a suitable natural polymer and adding a transdermal penetration enhancer to obtain the compound traditional Chinese medicine liposome gel patch. Animal experimental studies have shown that the patch prepared using the process of this invention can effectively alleviate the symptoms of early-onset ovarian insufficiency and slow down the rate of follicular atresia in the ovary.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical preparations, specifically relating to a compound traditional Chinese medicine liposome gel patch for treating early-onset ovarian insufficiency and its preparation method. Background Technology

[0002] Influenced by factors such as work, life, and social pressures, drug abuse, and unhealthy dietary habits, the prevalence of infertility among women of childbearing age is increasing year by year and showing a trend towards affecting younger women. Currently, the prevalence of infertility is relatively high in my country. Premature ovarian insufficiency (POI) caused by premature ovarian failure is a major factor leading to infertility. Its main cause is the decline in ovarian function leading to excessively high levels of follicle-stimulating hormone (FSH) (>10-15 mIU / L), excessively low levels of estrogen (<50 pg / ml), and low progesterone levels. Under the influence of these hormonal levels, symptoms such as irregular menstruation and dysmenorrhea often occur, eventually developing into premature ovarian insufficiency. Clinical manifestations of premature ovarian insufficiency include menstrual disorders, ovarian follicular atresia, and difficulty conceiving.

[0003] There are many compound traditional Chinese medicine formulas used to treat early-onset ovarian insufficiency. One of them, "Liu Zi Tang," is a proven formula developed by a renowned TCM doctor in Guangdong Province, who enjoys special government allowances. It was modified based on the ancient formula "Wu Zi Yan Zong Wan" and decades of clinical experience. It has a good therapeutic effect on early-onset ovarian insufficiency leading to infertility. "Liu Zi Tang" is composed of six basic herbs: wolfberry, privet fruit, rosehip, raspberry, dodder seed, and schisandra fruit. "Liu Zi Tang" is administered orally to patients using the traditional decoction method. Decoction is a relatively traditional method of processing Chinese medicine, increasing the interaction time between drug components, especially since some herbs are soaked or decocted beforehand. However, it is time-consuming, has a shorter shelf life after decoction, and is often bitter and has significant gastrointestinal irritation and toxic side effects. In order to overcome the shortcomings of traditional decoctions, oral preparations and topical formulations have begun to appear in compound Chinese medicines used to treat early-onset ovarian insufficiency.

[0004] CN109528980A discloses a traditional Chinese medicine composition for treating diseases of decreased ovarian reserve, and the method for preparing the traditional Chinese medicine composition into powder, tablet, capsule, granule, pill, compound, or decoction. The diseases of decreased ovarian reserve that can be treated are preferably ovarian reserve deficiency, premature ovarian insufficiency, premature ovarian failure, low ovarian response, and infertility and perimenopausal syndrome caused by the above diseases.

[0005] CN105943651A discloses a capsule, granule, tablet, oral liquid, compound or syrup preparation made from a traditional Chinese medicine composition for treating premature ovarian failure. The formulation has few medicinal ingredients, abundant raw materials, simple preparation process, and is environmentally friendly, showing good application prospects in the treatment of premature ovarian failure.

[0006] CN112138134A discloses a traditional Chinese medicine patch for treating gynecological diseases, which can be applied to different symptoms before and after ovulation. It can avoid the gastrointestinal irritation and toxic side effects of oral drugs, and has the dual therapeutic effects of transdermal drug absorption and stimulation of meridian acupoints. It has significant curative effect, low side effects, and is convenient to use.

[0007] Transdermal patches, manufactured using a transdermal delivery method, avoid the first-pass effect in the liver, enzymatic and hydrolytic degradation in the gastrointestinal tract, and gastrointestinal irritation. They offer advantages such as convenient administration and good patient compliance. Currently, many traditional Chinese medicine (TCM) compound prescriptions for treating early-onset ovarian insufficiency are administered transdermally using decocted TCM patches. However, complex TCM formulas like "Liu Zi Tang" contain a large number of active ingredients with poor water solubility. Decocted TCM patches have low skin penetration and low bioavailability, making them unsuitable for clinical treatment. The market needs a novel drug-loaded patch that can effectively encapsulate the TCM components, allowing "Liu Zi Tang" to fully exert its efficacy and effectively treat early-onset ovarian insufficiency. Summary of the Invention

[0008] Given the complexity of the active ingredients in the compound traditional Chinese medicine "Liuzi Tang" and the presence of a large number of poorly water-soluble active ingredients, the primary objective of this invention is to provide a method for preparing a compound traditional Chinese medicine liposome gel patch for treating early-onset ovarian insufficiency. Utilizing the advantages of liposomes' good drug-carrying capacity and high transdermal delivery effect, phospholipids and cholesterol biomolecules are selected to construct a nano-bilayer composite liposome. This allows the active ingredients of the traditional Chinese medicine to be simultaneously loaded into both the inner aqueous phase and the bilayer, achieving "dual drug loading" of both water-soluble and water-insoluble active ingredients to meet the drug loading requirements of the compound traditional Chinese medicine.

[0009] Another object of the present invention is to provide a compound traditional Chinese medicine liposome gel patch prepared by the above preparation method for treating early-onset ovarian insufficiency.

[0010] The objective of this invention is achieved through the following technical solution:

[0011] A method for preparing a compound traditional Chinese medicine liposome gel patch for treating early-onset ovarian insufficiency includes the following steps:

[0012] (1) The compound Chinese medicine raw materials are pulverized, their effective active ingredients are extracted, and the active ingredients are obtained by freeze drying.

[0013] (2) Phospholipids, cholesterol and surfactants are added to an alcohol solvent as the oil phase, and the active pharmaceutical ingredient is dissolved in a phosphate buffer as the aqueous phase. The oil phase and aqueous phase are mixed to form an emulsion, and the solvent is removed to obtain a colloidal state. Short-chain alcohols are then added for hydration, and the solution is filtered to obtain a drug-loaded liposome solution.

[0014] (3) After mixing the drug-loaded liposome solution with natural polymers and transdermal penetration enhancers, the pH is adjusted to form a gel, and a gel patch is obtained.

[0015] Preferably, the compound Chinese medicine raw materials in step (1) are wolfberry, dodder seed, raspberry, schisandra fruit, rosehip and privet fruit in a mass ratio of (1-3):(1-3):(1-2):(1-2):(1-2):(1-2); more preferably, the mass ratio of wolfberry, dodder seed, raspberry, schisandra fruit, rosehip and privet fruit is (1-3):(1-3):1:1:1:1.

[0016] Preferably, the extraction of effective active ingredients in step (1) is performed by ultrasonic extraction, wherein the material-to-liquid ratio is 1g:(10-20)ml, the temperature is 70-90℃, the extraction time is 1-2h, and the extraction solvent is at least one of water and ethanol; the number of extractions is ≥1.

[0017] More preferably, the material-to-liquid ratio is 1:20, the temperature is 80°C, the extraction time is 2 hours, and the extraction is performed twice.

[0018] Preferably, the freeze-drying temperature in step (1) is -70 to -30°C, and the time is 3 to 4 days.

[0019] Preferably, the phospholipid in step (2) is a natural phospholipid, specifically at least one of soybean lecithin and egg yolk lecithin.

[0020] Preferably, the surfactant in step (2) is at least one of Tween-80, cholate, deoxycholate, propylene glycol, and glycerol.

[0021] Preferably, the ratio of phospholipids, cholesterol, surfactants, active pharmaceutical ingredients and short-chain alcohols in step (2) is (4-12)g:(2-6)g:(1-2)g:(1-3)g:(40-120)ml; more preferably, it is 8g:2g:1g:1g:(40-120)ml.

[0022] Preferably, the alcohol solvent in step (2) is ethanol; the concentration of the phospholipid in the alcohol solvent is 10-30 mg / ml.

[0023] Preferably, the pH of the phosphate buffer solution in step (2) is 6 to 8; and the concentration of the active pharmaceutical ingredient in the phosphate buffer solution is 5 to 15 mg / ml.

[0024] Preferably, the volume ratio of the oil phase to the water phase in step (2) is (2-6):1.

[0025] Preferably, in step (2), the oil phase and water phase are mixed and then ultrasonicated for 20-30 minutes to form an emulsion; the solvent removal is carried out by rotary evaporation under reduced pressure at constant temperature.

[0026] Preferably, the short-chain alcohol in step (2) is at least one of ethanol, propylene glycol and glycerol.

[0027] Preferably, the short-chain alcohol in step (2) is added in solution form, wherein the volume concentration of the short-chain alcohol in the solution is 10-40%, and the solvent is a phosphate buffer solution with pH = 6-8.

[0028] Preferably, the hydration in step (2) is carried out by constant temperature shaking hydration and / or rotary evaporator depressurized rotary hydration; after hydration, ultrasonic mixing is performed for 2 to 4 minutes.

[0029] Preferably, the filtration in step (2) refers to passing the material through a 100nm microporous sieve.

[0030] Preferably, the natural polymer in step (3) is at least one of hyaluronic acid, gelatin, chitosan, alginate, fibrin, collagen and soft agar; the natural polymer is added in the form of a solution with a mass concentration of 0.5-3%, and the solvent is at least one of deionized water, acetic acid solution with a volume fraction of 1-2%, and sodium chloride solution with a mass fraction of 0.5-2%.

[0031] Preferably, the transdermal penetration enhancer in step (3) is at least one of menthol, dimethyl sulfoxide, borneol, angelica oil, clove oil and azone.

[0032] Preferably, the ratio of the drug-loaded liposome solution, natural polymer, and transdermal penetration enhancer in step (3) is 1 ml: 0.03-0.06 g: 0.05-0.15 ml.

[0033] Preferably, the pH adjuster used in step (3) is at least one of β-glycerophosphate, a calcium sulfate aqueous solution with a mass concentration of 1-3%, or a mixed solution of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC) and N-hydroxysuccinimide (NHS) with a mass ratio of 1-3:1.

[0034] The above preparation method yields a compound traditional Chinese medicine liposome gel patch for the treatment of early-onset ovarian insufficiency.

[0035] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0036] (1) This invention loads compound Chinese medicine into surface-modified flexible liposomes, especially by introducing short-chain alcohols into the phospholipid bilayer and adding active ingredients to modify the surface of the liposomes, which can effectively increase the encapsulation rate of single drugs and the types of drugs encapsulated, and has a good encapsulation effect on "Liuzitang".

[0037] (2) The patch described in this invention can promote the functional recovery of premature ovarian failure, including promoting the secretion of estradiol by the ovaries, reducing the level of follicle-stimulating hormone in the body, and increasing the index of ovaries, uterus and kidneys, thereby effectively treating premature ovarian insufficiency. Attached Figure Description

[0038] Figure 1 This is a schematic diagram of the structure of the "Liuzitang" nanobilayer composite liposome prepared in this invention;

[0039] Figure 2 The liposome particle size diagrams are for Examples 1-3 and Comparative Examples 1-3.

[0040] Figure 3 Standard curves for hyperoside, privetin, and schisandrol A were prepared.

[0041] Figure 4 The ovarian, uterine, and kidney indices of rats in each group;

[0042] Figure 5 The E2 content in the serum of rats in each group;

[0043] Figure 6 The FSH content of rats in each group is shown. Detailed Implementation

[0044] The present invention will be further described in detail below with reference to the embodiments and accompanying drawings, but the implementation of the present invention is not limited thereto.

[0045] Unless otherwise specified in the embodiments of this invention, the conditions shall be performed according to conventional conditions or conditions recommended by the manufacturer. All raw materials and reagents used, unless otherwise specified, are commercially available conventional products.

[0046] Example 1

[0047] The following herbs were mixed and pulverized using a pulverizer: wolfberry, dodder seed, raspberry, schisandra fruit, rosehip, and privet fruit (mass ratio 1.5:1.5:1:1:1:1). The pulverized active ingredient solid particles were dissolved in a 75% ethanol solution at a solid-liquid ratio of 1:10 g / ml. The mixture was ultrasonically vibrated at 500 W for 1 hour in a 70℃ water bath. After filtration, the residue was dissolved in the same volume of deionized water as the 75% ethanol solution for a second extraction (ultrasonic vibration at 500 W for 1 hour in a 70℃ water bath). The residue was then filtered, and the two filtrates were collected and freeze-dried to obtain the active ingredient solid particles.

[0048] 0.2g of egg yolk lecithin, 0.05g of cholesterol, and 0.025g of Tween-80 were dissolved in 10ml of anhydrous ethanol as the oil phase. Separately, 0.025g of the active pharmaceutical ingredient solid particles were dissolved in 3ml of phosphate buffer solution with pH 7.4 as the aqueous phase. The aqueous phase was added to the oil phase, and the mixture was sonicated for 30min to form a homogeneous emulsion. The solvent was then removed by rotary evaporation under reduced pressure at a constant temperature until a colloidal state was reached. 10ml of phosphate buffer solution containing 20% ​​(v / v) glycerol (pH 7.4) was added, and the mixture was hydrated by rotary evaporation at a constant temperature for 30min. The mixture was then sonicated in a water bath for 4min and extruded through a 100nm pore size filter membrane to obtain the drug-loaded liposome solution.

[0049] 1 g of chitosan was dissolved in 100 ml of 2% acetic acid aqueous solution to prepare a 1% chitosan solution. 1 volume part of liposome solution was mixed with 4 volume parts of the 1% chitosan solution. Then, 1% (v / v) menthol was added to the total solution of the liposome solution and chitosan solution. Finally, an appropriate amount of β-glycerophosphate was added to adjust the solution into a gel to obtain the patch.

[0050] Example 2

[0051] The following herbs were mixed and pulverized using a pulverizer: wolfberry, dodder seed, raspberry, schisandra fruit, rosehip, and privet fruit (mass ratio 1.5:1.5:1:1:1:1). The pulverized active ingredient solid particles were dissolved in a 75% ethanol solution at a solid-liquid ratio of 1:10 g / ml. The mixture was ultrasonically vibrated at 500 W for 1 hour in a 70℃ water bath. After filtration, the residue was dissolved in the same volume of deionized water as the 75% ethanol solution for a second extraction (ultrasonic vibration at 500 W for 1 hour in a 70℃ water bath). The residue was then filtered, and the two filtrates were collected and freeze-dried to obtain the active ingredient solid particles.

[0052] 0.2g of egg yolk lecithin, 0.05g of cholesterol, and 0.025g of Tween-80 were dissolved in 10ml of anhydrous ethanol as the oil phase. Separately, 0.025g of the active pharmaceutical ingredient solid particles were dissolved in 3ml of phosphate buffer solution with a pH of 7.4 as the aqueous phase. The aqueous phase was added to the oil phase, and the mixture was sonicated for 30min to form a homogeneous emulsion. The solvent was then removed by rotary evaporation under reduced pressure at a constant temperature until a colloidal state was reached. 10ml of phosphate buffer solution containing 10% (v / v) glycerol was added, and the mixture was hydrated by rotary evaporation at a constant temperature for 30min. The mixture was then sonicated in a water bath for 4min and extruded through a 100nm pore size filter membrane to obtain the drug-loaded liposome solution.

[0053] 1 g of chitosan was dissolved in 100 ml of 2% acetic acid aqueous solution to prepare a 1% chitosan solution. 1 volume part of liposome solution was mixed with 4 volume parts of the 1% chitosan solution. Then, 1% (v / v) menthol was added to the total solution of the liposome solution and chitosan solution. Finally, an appropriate amount of β-glycerophosphate was added to adjust the solution into a gel to obtain the patch.

[0054] Example 3

[0055] The following herbs were mixed and pulverized using a pulverizer: wolfberry, dodder seed, raspberry, schisandra fruit, rosehip, and privet fruit (mass ratio 1.5:1.5:1:1:1:1). The pulverized active ingredient solid particles were dissolved in a 75% ethanol solution at a solid-liquid ratio of 1:10 g / ml. The mixture was ultrasonically vibrated at 500 W for 1 hour in a 70℃ water bath. After filtration, the residue was dissolved in the same volume of deionized water as the 75% ethanol solution for a second extraction (ultrasonic vibration at 500 W for 1 hour in a 70℃ water bath). The residue was then filtered, and the two filtrates were collected and freeze-dried to obtain the active ingredient solid particles.

[0056] 0.2g of egg yolk lecithin, 0.05g of cholesterol, and 0.025g of Tween-80 were dissolved in 10ml of anhydrous ethanol as the oil phase. Separately, 0.025g of the active pharmaceutical ingredient solid particles were dissolved in 3ml of phosphate buffer solution with a pH of 7.4 as the aqueous phase. The aqueous phase was added to the oil phase, and the mixture was sonicated for 30min to form a homogeneous emulsion. The solvent was then removed by rotary evaporation under reduced pressure at a constant temperature until a colloidal state was reached. 10ml of phosphate buffer solution containing 30% (v / v) glycerol was added, and the mixture was hydrated by rotary evaporation at a constant temperature for 30min. The mixture was then sonicated in a water bath for 4min and extruded through a 100nm pore size filter membrane to obtain the drug-loaded liposome solution.

[0057] 1 g of chitosan was dissolved in 100 ml of 2% acetic acid aqueous solution to prepare a 1% chitosan solution. 1 volume part of liposome solution was mixed with 4 volume parts of the 1% chitosan solution. Then, 1% (v / v) menthol was added to the total solution of the liposome solution and chitosan solution. An appropriate amount of β-glycerophosphate was added to adjust the mixture into a gel to obtain the patch.

[0058] Comparative Example 1 (without short-chain alcohols)

[0059] The following herbs were mixed and pulverized using a pulverizer: wolfberry, dodder seed, raspberry, schisandra fruit, rosehip, and privet fruit (mass ratio 1.5:1.5:1:1:1:1). The pulverized active ingredient solid particles were dissolved in a 75% ethanol solution at a solid-liquid ratio of 1:10 g / ml. The mixture was ultrasonically vibrated at 500 W for 1 hour in a 70℃ water bath. After filtration, the residue was dissolved in the same volume of deionized water as the 75% ethanol solution for a second extraction (ultrasonic vibration at 500 W for 1 hour in a 70℃ water bath). The residue was then filtered, and the two filtrates were collected and freeze-dried to obtain the active ingredient solid particles.

[0060] 0.2g of egg yolk lecithin, 0.05g of cholesterol, and 0.025g of Tween-80 were dissolved in 10ml of anhydrous ethanol as the oil phase. Separately, 0.025g of the active pharmaceutical ingredient solid particles were dissolved in 3ml of phosphate buffer solution with a pH of 7.4 as the aqueous phase. The aqueous phase was added to the oil phase, and the mixture was sonicated for 30min to form a homogeneous emulsion. The solvent was then removed by rotary evaporation under reduced pressure at a constant temperature until a colloidal state was reached. 10ml of phosphate buffer solution was added, and the mixture was hydrated by rotary evaporation at a constant temperature for 30min. The mixture was then sonicated in a water bath for 4min. The drug-loaded liposome solution was obtained by extrusion through a filter membrane with a pore size of 100nm.

[0061] 1 g of chitosan was dissolved in 100 ml of 2% acetic acid aqueous solution to prepare a 1% chitosan solution. 1 volume part of liposome solution was mixed with 4 volume parts of the 1% chitosan solution. Then, 1% (v / v) menthol was added to the total solution of the liposome solution and chitosan solution. Finally, an appropriate amount of β-glycerophosphate was added to adjust the mixture into a gel to obtain the patch.

[0062] Comparative Example 2 (short-chain alcohols were added at different times)

[0063] The following herbs were mixed and pulverized using a pulverizer: wolfberry, dodder seed, raspberry, schisandra fruit, rosehip, and privet fruit (mass ratio 1.5:1.5:1:1:1:1). The pulverized active ingredient solid particles were dissolved in a 75% ethanol solution at a solid-liquid ratio of 1:10 g / ml. The mixture was ultrasonically vibrated at 500 W for 1 hour in a 70℃ water bath. After filtration, the residue was dissolved in the same volume of deionized water as the 75% ethanol solution for a second extraction (ultrasonic vibration at 500 W for 1 hour in a 70℃ water bath, followed by filtration). The filtrates from both extractions were collected and freeze-dried to obtain the active ingredient solid particles.

[0064] 0.2 g of egg yolk lecithin, 0.05 g of cholesterol, and 0.025 g of Tween-80 were dissolved in 10 ml of anhydrous ethanol as the oil phase. Separately, 0.025 g of the active pharmaceutical ingredient solid particles were dissolved in 3 ml of phosphate buffer (pH 7.4) containing 66.67% (v / v) glycerol as the aqueous phase. The aqueous phase was added to the oil phase, and the mixture was sonicated for 30 min to form a homogeneous emulsion. The solvent was then removed by rotary evaporation under reduced pressure at a constant temperature until a colloidal state was reached. 10 ml of phosphate buffer solution with pH 7.4 was added, and the mixture was hydrated by rotary evaporation at a constant temperature for 30 min. The mixture was then sonicated in a water bath for 4 min. The drug-loaded liposome solution was obtained by extrusion through a 100 nm pore size filter membrane.

[0065] 1 g of chitosan was dissolved in 100 ml of 2% acetic acid aqueous solution to prepare a 1% chitosan solution. 1 volume part of liposome solution was mixed with 4 volume parts of the 1% chitosan solution. Then, 1% (v / v) menthol was added to the total solution of the liposome solution and chitosan solution. Finally, an appropriate amount of β-glycerophosphate was added to adjust the pH to form a gel, thus obtaining the patch.

[0066] Comparative Example 3 (no surfactant added and the timing of addition of short-chain alcohols differs)

[0067] The following herbs were mixed and pulverized using a pulverizer: wolfberry, dodder seed, raspberry, schisandra fruit, rosehip, and privet fruit (mass ratio 1.5:1.5:1:1:1:1). The pulverized active ingredient solid particles were dissolved in a 75% ethanol solution at a solid-liquid ratio of 1:10 g / ml. The mixture was ultrasonically vibrated at 500 W for 1 hour in a 70℃ water bath. After filtration, the residue was dissolved in the same volume of deionized water as the 75% ethanol solution for a second extraction (ultrasonic vibration at 500 W for 1 hour in a 70℃ water bath). The residue was then filtered, and the two filtrates were collected and freeze-dried to obtain the active ingredient solid particles.

[0068] 0.2g of egg yolk lecithin and 0.05g of cholesterol were dissolved in 10ml of anhydrous ethanol as the oil phase. Separately, 0.025g of the active pharmaceutical ingredient solid particles were dissolved in 3ml of phosphate buffer containing 66.67% glycerol (pH 7.4) as the aqueous phase. The aqueous phase was added to the oil phase, and the mixture was sonicated for 30min to form a homogeneous emulsion. The solvent was then removed by rotary evaporation under reduced pressure at a constant temperature until a colloidal state was reached. 10ml of pH 7.4 buffer solution was added, and the mixture was hydrated by rotary evaporation at a constant temperature for 30min. The mixture was then sonicated in a water bath for 4min and extruded through a 100nm pore size filter membrane to obtain the drug-loaded liposome solution.

[0069] 1 g of chitosan was dissolved in 100 ml of 2% acetic acid aqueous solution to prepare a 1% chitosan solution. 1 volume part of liposome solution was mixed with 4 volume parts of the 1% chitosan solution. Then, 1% (v / v) menthol was added to the total solution of the liposome solution and chitosan solution. Finally, an appropriate amount of β-glycerophosphate was added to adjust the mixture into a gel to obtain the patch.

[0070] Comparative Example 4 (Liposome-free solution)

[0071] The following herbs were mixed and pulverized using a pulverizer: wolfberry, dodder seed, raspberry, schisandra fruit, rosehip, and privet fruit (mass ratio 1.5:1.5:1:1:1:1). The pulverized active ingredient solid particles were dissolved in a 75% ethanol solution at a solid-liquid ratio of 1:10 g / ml. The mixture was ultrasonically vibrated at 500 W for 1 hour in a 70℃ water bath. After filtration, the residue was dissolved in the same volume of deionized water as the 75% ethanol solution for a second extraction (ultrasonic vibration at 500 W for 1 hour in a 70℃ water bath). The residue was then filtered, and the two filtrates were collected and freeze-dried to obtain the active ingredient solid particles.

[0072] Dissolve 0.025g of the active pharmaceutical ingredient solid particles in 10ml of phosphate buffer solution with pH 7.4 to prepare a phosphate solution of the active pharmaceutical ingredient.

[0073] 1 g of chitosan was dissolved in 100 ml of 2% acetic acid aqueous solution to prepare a 1% chitosan solution. 1 volume part of the active pharmaceutical ingredient phosphate solution was mixed with 4 volume parts of the 1% chitosan solution. Then, 1% (v / v) menthol was added to the total solution of the mixture of the active pharmaceutical ingredient phosphate solution and chitosan solution. Finally, an appropriate amount of β-glycerophosphate was added to adjust the mixture into a gel to obtain the patch.

[0074] Experimental Example 1: Liposome Encapsulation Efficiency Experiment

[0075] The extracted and freeze-dried solid particles of the active pharmaceutical ingredient were dissolved in methanol and analyzed by high performance liquid chromatography to establish a reference spectrum for "Liuzitang".

[0076] The testing method is as follows:

[0077] The instrument was a Shimadzu LAD-20; the mobile phase was acetonitrile-0.4% phosphoric acid aqueous solution; the column was a C18 reverse-phase column; the column temperature was 40℃; the flow rate was 1.0 ml / min; the detector wavelength was 254 nm; the elution gradient was 0-5 min, 5-15% acetonitrile, 5-10 min, 15-17% acetonitrile, 10-25 min, 17% acetonitrile, 25-35 min, 17-26% acetonitrile, and 35-60 min, 26-56% acetonitrile.

[0078] Liposomes and drugs were separated using ultrafiltration centrifugation. The separated drugs were then analyzed by high-performance liquid chromatography (HPLC). The encapsulation efficiency of various drugs, the similarity between the encapsulated drug chromatogram and the reference spectrum, and the number of common peaks were calculated.

[0079] The formula for calculating the encapsulation ratio is:

[0080] (W 总 -W 游 ) / W 总 ×100%

[0081] Among them W 总 W represents the total drug content in the liposome solution. 游 This refers to the amount of free drug in the isolated liposome solution.

[0082] Table 1. Encapsulation efficiency (%) of liposomes in the examples and comparative examples

[0083] Hypericin Specific privetin Schisandra chinensis alcohol A Example 1 99.89 97.56 96.45 Example 2 98.76 95.24 93.59 Example 3 98.45 98.31 98.29 Comparative Example 1 83.34 89.77 85.21 Comparative Example 2 89.17 91.47 88.23 Comparative Example 3 78.34 76.46 83.50

[0084] Table 2. Similarity and number of common peaks of liposome-encapsulated drugs between the examples and comparative examples.

[0085] Similarity % Total number of peaks Example 1 96.42 36 Example 2 96.61 36 Example 3 95.76 36 Comparative Example 1 86.73 33 Comparative Example 2 93.53 34 Comparative Example 3 90.67 31

[0086] Experiment Example 2: Lipid Physical Properties Experiment

[0087] The particle size and polydispersity index of liposomes were measured using a laser particle size analyzer.

[0088] Table 3. Similarity and number of common peaks of liposome-encapsulated drugs between the examples and comparative examples.

[0089] Liposome particle size / nm polydispersion coefficient Example 1 94.76 0.080 Example 2 94.94 0.078 Example 3 94.22 0.086 Comparative Example 1 110.0 0.093 Comparative Example 2 103.4 0.091 Comparative Example 3 123.2 0.128

[0090] As can be seen from Table 1-2, the liposome encapsulation efficiency and encapsulation effect are related to the proportion of glycerol in the hydration solution, surfactants, etc. In the liposome preparation prepared by the technical solution protected by the present invention, the liposome encapsulation efficiency and the similarity of the encapsulated drug are both above 90%. The encapsulation efficiency of the best technical solution (Example 1) reaches 96%, the similarity reaches 96.42%, and the number of common peaks is 36, indicating that the prepared liposomes have excellent encapsulation efficiency and encapsulation effect on "Liuzitang", and the liposome particle size is smaller and the particle size distribution is uniform.

[0091] Experimental Example 3 Pharmacological Experiment of the Patch

[0092] The following is the efficacy research situation of the present invention:

[0093] The inventors carried out relevant pharmacodynamic experimental studies to prove the efficacy of the compound traditional Chinese medicine liposome gel patch of the present invention in the treatment of premature ovarian insufficiency. This experiment has been approved by the ethics committee. The patches selected in the following pharmacodynamic experiments are the drugs obtained from the representative formulations of the present invention; the preparations obtained from other embodiments included in the present invention have the same or similar experimental results, but due to space limitations, they are not listed one by one here.

[0094] 1. Experimental Animals

[0095] 32 SPF-grade female SD rats at 3 weeks of age, each rat weighing about (140 ± 10) g, were purchased from the Animal Experiment Center of South China University of Technology (certificate number NO. 430727211102821752, license number SYXK (Guangdong) 2017-0178). They were quarantined and raised in the Animal Experiment Center of South China University of Technology for 1 week, fed with sterilized pellet feed, allowed to feed freely, under standard lighting, at a room temperature of 20-25 °C, and a relative humidity of 45%-55%. [[ID=***]] [[ID=***]]

[0096] Experimental Methods

[0097] Animal Modeling and Drug Administration

[0098] The 32 female SD rats were randomly divided into four groups (control group, Example 1 group, Comparative Example 1 group, Comparative Example 4 group), and each group was intraperitoneally injected with VCD solution at a dose of 160 mg / (kg·d) for 15 consecutive days. The chemical substance 4-vinylcyclohexene dioxide (VCD) has been described as capable of inducing accelerated depletion of primordial follicles and primary follicles in female rodents. Therefore, treating rats with this compound in the early stage of life can induce ovarian function decline before central changes occur.

[0099] Two weeks after modeling, the estrous cycle of rats was observed, and rats with successful modeling were selected for further experiments. A 2% sodium pentobarbital solution was prepared with distilled water and administered intraperitoneally at a dose of 40 mg / kg to anesthetize the rats. The skin on the rat abdomen was prepared and the hair removed using a pet clipper, followed by depilatory cream. The control group received a blank patch, Example 1 group received the Example 1 patch, Comparative Example 1 group received the Comparative Example 1 patch, and Comparative Example 4 group received the Comparative Example 4 patch. Administration was carried out for 8 hours daily for 28 days.

[0100] Specimen Collection

[0101] After modeling and administration, the rats were weighed. The rats were anesthetized with sodium pentobarbital intraperitoneally and 2 ml of blood was collected through the abdominal aorta using a disposable blood collection needle. The blood sample was then centrifuged at 4°C and 1500 rpm / min for 15 min. After centrifugation, the upper serum layer was collected and transferred to a new centrifuge tube, which was then frozen at -20°C for later use.

[0102] Bilateral ovaries, kidneys, and uteruses of each group of animals were removed by laparotomy. After removing the adipose tissue attached to the organs, they were weighed. After weighing, each ovary, uterus, and kidney was fixed by immersing in 4% paraformaldehyde.

[0103] detection indicators

[0104] Weight changes and ovarian and uterine weight and ovarian and uterine index

[0105] Record the body weight of rats in each group after the administration of the drug.

[0106] During specimen collection, the bilateral ovaries, kidneys, and uterus of the rats were removed, and their wet weight was recorded. The ovarian index, uterine index, and kidney index were calculated as follows: Ovarian index (mg / g) = ovarian mass (mg) / body mass (g) × 100%; Uterine index (mg / g) = uterine mass (mg) / body mass (g) × 100%; Kidney index (mg / g) = kidney mass (mg) / body mass (g) × 100%.

[0107] serum hormones

[0108] After thawing the collected frozen serum, the concentrations of E2 and FSH in the serum were measured according to the requirements of the ELISA kit.

[0109] Experimental results

[0110] Table 4 Ovarian, uterine, and kidney indices of rats in each group

[0111]

[0112]

[0113] Depend on Figure 3-5 As shown in Table 4, the compound traditional Chinese medicine liposome gel patch of the present invention has a good effect on the treatment of early-onset ovarian insufficiency: it has a good promoting effect on the recovery of atrophied ovarian and uterine tissues, and at the same time, it also improves the kidney index to a certain extent and promotes the recovery of the level of related hormones in the kidney.

[0114] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A method for preparing a compound traditional Chinese medicine liposome gel patch for treating early-onset ovarian insufficiency, characterized in that, Includes the following steps: (1) The compound Chinese medicine raw materials are pulverized, their effective active ingredients are extracted, and the active ingredients are obtained by freeze drying. (2) Phospholipids, cholesterol and surfactants are added to an alcohol solvent as the oil phase, and the active pharmaceutical ingredient is dissolved in a phosphate buffer as the aqueous phase. The oil phase and aqueous phase are mixed to form an emulsion, and the solvent is removed to obtain a colloidal state. Short-chain alcohols are then added for hydration, and the solution is filtered to obtain a drug-loaded liposome solution. (3) After mixing the drug-loaded liposome solution with natural polymers and transdermal penetration enhancers, the pH is adjusted to form a gel, and a gel patch is obtained; The compound Chinese medicine raw materials mentioned in step (1) are wolfberry, dodder seed, raspberry, schisandra fruit, rosehip and privet fruit in a mass ratio of (1-3):(1-3):(1-2):(1-2):(1-2):(1-2); The ratio of phospholipids, cholesterol, surfactants, active pharmaceutical ingredients and short-chain alcohols in step (2) is (4-12)g:(2-6)g:(1-2)g:(1-3)g:(40-120)ml; the surfactant is at least one of Tween-80 cholate, deoxycholate, propylene glycol and glycerol. The ratio of the drug-loaded liposome solution, natural polymer, and transdermal penetration enhancer in step (3) is 1 ml: 0.03-0.06 g: 0.05-0.15 ml.

2. The preparation method of the compound traditional Chinese medicine liposome gel patch for treating early-onset ovarian insufficiency according to claim 1, characterized in that, The short-chain alcohol in step (2) is at least one of ethanol, propylene glycol and glycerol.

3. The preparation method of the compound traditional Chinese medicine liposome gel patch for treating early-onset ovarian insufficiency according to claim 1, characterized in that, The phospholipids mentioned in step (2) are natural phospholipids; the natural phospholipids are at least one of soybean lecithin and egg yolk lecithin.

4. The preparation method of the compound traditional Chinese medicine liposome gel patch for treating early-onset ovarian insufficiency according to claim 1, characterized in that, The natural polymer mentioned in step (3) is at least one of hyaluronic acid, gelatin, chitosan, alginate, fibrin, collagen and soft agar; The transdermal penetration enhancer in step (3) is at least one of menthol, dimethyl sulfoxide, borneol, angelica oil, clove oil and azone; The pH adjuster used in step (3) is at least one of β-glycerophosphate, a calcium sulfate aqueous solution with a mass concentration of 1-3%, or a mixed solution of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and N-hydroxysuccinimide with a mass ratio of 1-3:

1.

5. The preparation method of the compound traditional Chinese medicine liposome gel patch for treating early-onset ovarian insufficiency according to claim 1, characterized in that, In step (2), the short-chain alcohol is added in solution form, wherein the volume concentration of the short-chain alcohol in the solution is 10-40%, and the solvent is a phosphate buffer solution with pH = 6-8. The hydration in step (2) is carried out by constant temperature shaking hydration and / or rotary evaporator depressurized rotary hydration; after hydration, ultrasonic mixing is performed for 2 to 4 minutes.

6. The preparation method of the compound traditional Chinese medicine liposome gel patch for treating early-onset ovarian insufficiency according to claim 1, characterized in that, The alcohol solvent in step (2) is ethanol; the concentration of the phospholipid in the alcohol solvent is 10-30 mg / ml; The pH of the phosphate buffer solution in step (2) is 6-8; the concentration of the active pharmaceutical ingredient in the phosphate buffer solution is 5-15 mg / ml. The volume ratio of the oil phase to the water phase in step (2) is (2-6):

1.

7. The preparation method of the compound traditional Chinese medicine liposome gel patch for treating early-onset ovarian insufficiency according to claim 1, characterized in that, The effective active ingredient in step (1) is extracted by ultrasonic extraction, wherein the material-to-liquid ratio is 1g:(10-20)ml, the temperature is 70-90℃, the extraction time is 1-2h, and the extraction solvent is at least one of water and ethanol; the number of extractions is ≥1.

8. The preparation method of the compound traditional Chinese medicine liposome gel patch for treating early-onset ovarian insufficiency according to claim 1, characterized in that, The natural polymer in step (3) is added in the form of a solution with a mass concentration of 0.5-3%, and the solvent is at least one of deionized water, acetic acid solution with a volume fraction of 1-2%, and sodium chloride solution with a mass fraction of 0.5-2%.

9. The preparation method of the compound traditional Chinese medicine liposome gel patch for treating early-onset ovarian insufficiency according to claim 1, characterized in that, In step (2), the oil phase and water phase are mixed and then ultrasonicated for 20-30 minutes to form an emulsion; the solvent removal is carried out by rotary evaporation under reduced pressure at constant temperature. The filtration in step (2) refers to passing the material through a 100nm microporous sieve.

10. A compound traditional Chinese medicine liposome gel patch for treating early-onset ovarian insufficiency, prepared by the method according to any one of claims 1 to 9.

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