Isoliquiritigenin-loaded drug-loaded liposome as well as preparation method and application thereof

Abstract

The invention provides an isoliquiritigenin-loaded drug-loaded liposome as well as a preparation method and application thereof, and relates to the technical field of drugs. The drug-loaded liposome is prepared from the following raw materials in parts by weight: 20-50 parts of lecithin, 6-15 parts of glycyrrhetinic acid and 2-10 parts of isoliquiritigenin, and can be used as a novel drug for treating androgenetic alopecia.

Description

Technical Field

[0001] The present invention relates to the field of pharmaceutical technology, in particular to a drug-loaded liposome loaded with isoliquiritigenin, a preparation method and application thereof. Background Art

[0002] The following statements merely provide background information related to the present disclosure and do not necessarily constitute prior art.

[0003] Androgenetic alopecia (AGA) is the most common form of hair loss worldwide. It is caused by excessive androgen secretion and is characterized by progressive terminal hair loss after puberty, accompanied by elevated levels of inflammation and oxidative stress. The severity of androgenetic alopecia increases with age, with a distribution pattern unique to both sexes. Male pattern hair loss (MPHL) is characterized by gradual thinning of hair, primarily affecting the vertex and forehead, with a receding hairline. Female pattern hair loss (FPHL) is characterized by diffuse hair loss and thinning, typically sparing the hairline.

[0004] Despite its high prevalence, AGA can be challenging to treat due to its chronic nature and the interplay of genetic and environmental factors. Currently, the main medications used to treat AGA are topical minoxidil and oral finasteride. However, minoxidil is associated with side effects such as headache, hirsutism, irritation, and contact dermatitis; while finasteride may cause sexual dysfunction, gynecomastia, and mood changes.

[0005] As drug carriers, liposomes not only promote transdermal absorption but also maintain high drug concentrations in the skin. More importantly, liposomes possess a pro-hair follicle affinity, enabling targeted accumulation of drug-loaded liposomes in hair follicles. Cholesterol is a key component of liposomes, inserted perpendicular to the plane of the lipid bilayer. The C-3 hydroxyl group forms hydrogen bonds with the polar headgroups of phospholipids, maintaining the stability of the liposome phospholipid bilayer. However, cholesterol is susceptible to oxygen, light, and metals, generating cholesterol oxidation products during processing and storage that exhibit certain cytotoxic, mutagenic, and potentially carcinogenic properties.

[0006] Therefore, it is urgent to screen a new safe and effective ingredient and preparation to improve AGA.

[0007] In view of this, the present invention is proposed. Summary of the Invention

[0008] The purpose of the present invention is to provide a drug-loaded liposome loaded with isoliquiritigenin, so as to provide a new drug for treating androgenic alopecia.

[0009] In order to solve the above technical problems, the present invention adopts the following technical solutions: In a first aspect, a drug-loaded liposome loaded with isoliquiritigenin is provided, wherein the drug-loaded liposome is prepared from raw materials comprising 20 to 50 parts by weight of lecithin, 6 to 15 parts by weight of glycyrrhetinic acid, and 2 to 10 parts by weight of isoliquiritigenin.

[0010] In a second aspect, a method for preparing drug-loaded liposomes loaded with isoliquiritigenin is provided, the preparation method comprising dissolving lecithin, glycyrrhetinic acid and isoliquiritigenin in an organic phase, rotary evaporating to form a dispersed film, injecting into an aqueous phase, incubating and then sonicating to obtain the drug-loaded liposomes; The weight proportion of lecithin is 20 to 50 parts by weight, the weight proportion of glycyrrhetinic acid is 6 to 15 parts by weight, and the weight proportion of isoliquiritigenin is 2 to 10 parts by weight.

[0011] In a third aspect, the invention provides the use of the drug-loaded liposome described in the first aspect or the preparation method described in the second aspect in any of the following: (I) Preparation of medicaments for preventing and / or treating androgenic alopecia; (II) preparing a drug for alleviating inflammation; (III) Preparation of drugs for antioxidant activity; (IV) preparing a drug for inhibiting IL-6 and / or TNF-α; (V) Inhibition of IL-6 and / or TNF-α for non-diagnostic and non-therapeutic purposes.

[0012] In a fourth aspect, a pharmaceutical composition is also provided, comprising the drug-loaded liposomes described in the first aspect, or the drug-loaded liposomes prepared by the preparation method described in the second aspect.

[0013] Compared with the prior art, the present invention has the following beneficial effects: The drug-loaded liposomes provided by the present invention use lecithin and glycyrrhetinic acid as carriers and are loaded with isoliquiritigenin. Due to the characteristics of the liposomes, the drug-loaded liposomes have hair follicle targeting and can be directionally enriched in the hair follicle area. As an active ingredient of the drug, isoliquiritigenin can inhibit the conversion of testosterone into dihydrotestosterone by exerting its phytoestrogen properties, antagonizing androgens to a certain extent, thereby activating hair follicles, causing the hair follicles to transition from the resting phase to the growth phase, and promoting hair growth. In addition, the water solubility, hair follicle targeting and stability of isoliquiritigenin are enhanced by liposome encapsulation technology, which promotes the hair coverage area, hair length and hair follicle number of androgenic alopecia mice. The drug-loaded liposomes provided by the present invention also have better anti-inflammatory and antioxidant effects. This application verifies the role of drug-loaded liposomes loaded with isoliquiritigenin in the prevention and treatment of androgenic alopecia, broadens the application range of isoliquiritigenin, and also provides new candidate drugs for the prevention and treatment of androgenic alopecia diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the specific embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0015] Figure 1 1 is a particle size distribution diagram of a liposome sample in pure water having a weight ratio of lecithin, glycyrrhetinic acid, and isoliquiritigenin of 20:15:2 in Example 2; Figure 2 1 is a particle size distribution diagram of a liposome sample in pure water having a weight ratio of lecithin, glycyrrhetinic acid, and isoliquiritigenin of 30:9:5 in Example 2; Figure 3 1 is a particle size distribution diagram of a liposome sample in pure water having a weight ratio of lecithin, glycyrrhetinic acid, and isoliquiritigenin of 30:9:10 in Example 2; Figure 4 1 is a particle size distribution diagram of a liposome sample in pure water in which the weight ratio of lecithin, glycyrrhetinic acid and isoliquiritigenin is 50:15:5 in Example 2; Figure 5 1 is a particle size distribution diagram of a liposome sample in pure water in which the weight ratio of lecithin, glycyrrhetinic acid and isoliquiritigenin is 50:15:10 in Example 2; Figure 6 1 is a particle size distribution diagram of a liposome sample in pure water having a weight ratio of 50:6:10 among lecithin, glycyrrhetinic acid, and isoliquiritigenin in Example 2; Figure 7 IL-6 concentrations in the cell supernatant of each experimental group in Example 3, wherein NC is the blank control group, LPS is the LPS-induced inflammation group, GA-ISL-Lipo is the glycyrrhetinic acid liposome-loaded isoliquiritigenin administration group, and CHOL-ISL-Lipo is the cholesterol liposome-loaded isoliquiritigenin administration group; Figure 8 is the TNF-α concentration of the cell supernatant of each experimental group in Example 3, wherein NC is the blank control group, LPS is the LPS-induced inflammation group, GA-ISL-Lipo is the glycyrrhetinic acid liposome-loaded isoliquiritigenin administration group, and CHOL-ISL-Lipo is the cholesterol liposome-loaded isoliquiritigenin administration group; Figure 9 is the DPPH inhibition rate of the GA-ISL-Lipo group and the CHOL-ISL-Lipo group in Example 3, wherein GA-ISL-Lipo is the glycyrrhetinic acid liposome administration group loaded with isoliquiritigenin, and CHOL-ISL-Lipo is the cholesterol liposome administration group loaded with isoliquiritigenin; Figure 10 This is a graph showing the effect of glycyrrhetinic acid liposomes loaded with isoliquiritigenin on hair growth in mice with testosterone-induced androgenic alopecia in Example 5, wherein NC is a blank control group, AGA is a testosterone-induced androgenic alopecia group, AGA+GA-ISL-Lipo is a glycyrrhetinic acid liposomes loaded with isoliquiritigenin treatment group, and AGA+MND is a 2% minoxidil treatment group for androgenic alopecia (positive control group); A shows the back skin condition of mice in each group on the first day of administration 28 days after subcutaneous injection of testosterone; B shows the back hair growth condition of mice on the 7th day of administration; C shows the back hair growth condition of mice on the 14th day of administration; and D shows the back hair growth condition of mice on the 21st day of administration. Figure 11 The figure shows the hair coverage rate of mice with androgenic alopecia promoted by glycyrrhizic acid liposomes loaded with isoliquiritigenin in Example 4, which shows the hair coverage on days 7, 14, and 21 of administration, respectively. NC is the blank control group, AGA is the testosterone-induced androgenic alopecia group, AGA+GA-ISL-Lipo is the androgenic alopecia treatment group loaded with isoliquiritigenin and glycyrrhizic acid liposomes, and AGA+MND is the androgenic alopecia treatment group with 2% minoxidil (positive control group). Figure 12 This is a statistical graph of hair length in mice with androgenic alopecia induced by isoliquiritigenin in Example 4, wherein NC is a blank control group, AGA is a testosterone-induced androgenic alopecia group, AGA+GA-ISL-Lipo is a glycyrrhetinic acid liposome-loaded with isoliquiritigenin treatment group, and AGA+MND is a 2% minoxidil treatment group for androgenic alopecia (positive control group); Figure 13 The figure shows the results of hematoxylin-eosin staining of hair follicle tissue of androgenic alopecia mice treated with isoliquiritigenin in Example 6, wherein NC is the blank control group, AGA is the testosterone-induced androgenic alopecia group, AGA+GA-ISL-Lipo is the androgenic alopecia treated group with isoliquiritigenin-loaded glycyrrhetinic acid liposomes, and AGA+MND is the androgenic alopecia treated group with 2% minoxidil (positive control group). DETAILED DESCRIPTION

[0016] The following will clearly and completely describe the technical solutions of the present invention in conjunction with the embodiments. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0017] In this document, unless otherwise specified, all implementation methods and preferred implementation methods mentioned herein can be combined with each other to form a new technical solution.

[0018] In this document, unless otherwise specified, all technical features and preferred features mentioned herein can be combined with each other to form a new technical solution.

[0019] Herein, unless otherwise specified, the components or preferred components involved can be combined with each other to form a new technical solution.

[0020] Unless otherwise specified, the numerical range "a-b" is an abbreviation for any combination of real numbers between a and b, where a and b are real numbers. For example, the numerical range "6-22" indicates that all real numbers between "6-22" are listed in this document, and "6-22" is simply an abbreviation for these numerical combinations.

[0021]

[0046] "Range" disclosed herein in the form of lower limits and upper limits can have one or more lower limits, and one or more upper limits, respectively.

[0022] Herein, unless otherwise indicated, the various reactions or process steps may be performed sequentially or in any non-sequential manner. Preferably, the reaction methods herein are performed sequentially.

[0023] As used herein, the term "inhibit" is used interchangeably with "reduce," "silence," "downregulate," "suppress," and other similar terms, and includes any level of inhibition. Inhibition can be assessed by a reduction in the absolute or relative level of one or more of these variables compared to a control level. The control level can be any type of control level used in the art, such as a pre-dose baseline level or a level determined from an untreated or control-treated subject, cell, or sample (e.g., a buffer-only control or an inert agent control).

[0024] As used herein, the terms "patient," "subject," or "individual" are used interchangeably and include humans or non-human animals, such as humans, monkeys, mice, rats, rabbits, donkeys, cows, horses, pigs, or dogs, or cells or tissues derived from humans or non-human animals.

[0025] As used herein, the terms "treat," "treat," or "ameliorate" are used interchangeably herein. These terms refer to an approach for obtaining beneficial or desired results, including but not limited to a therapeutic benefit. "Therapeutic benefit" means the eradication or amelioration of one or more physiological symptoms associated with the underlying disorder being treated.

[0026] As used herein, the terms "prevention" and "prevention" are used interchangeably to refer to an approach for obtaining beneficial or desired results, including but not limited to a prophylactic benefit. To obtain a "prophylactic benefit," an agent may be administered to a subject at risk for a particular disease, or to a subject reporting one or more physiological symptoms of a disease, even though a diagnosis of the disease may not have yet been made.

[0027] Herein, the terms "comprise" or "comprising" are intended to imply the inclusion of stated elements, integers or steps, but not the exclusion of any other elements, integers or steps.

[0028] Unless otherwise indicated, the professional and scientific terms used herein are the same as those familiar to those skilled in the art. In addition, any method or material similar or equivalent to the described content can also be applied to the present invention.

[0029] In a first aspect, a drug-loaded liposome loaded with isoliquiritigenin is provided, wherein the drug-loaded liposome comprises lecithin, glycyrrhetinic acid and isoliquiritigenin.

[0030] Isoliquiritegenin (ISL) is a biomarker component in licorice, exhibiting antioxidant and anti-inflammatory activities. The structure of ISL is shown in formula (I), and it is an estrogen analog.

[0031] Formula (I).

[0032] Licorice is a traditional Chinese medicine with phytoestrogen activity. ISL is one of the flavonoid components in licorice with phytoestrogen activity. Androgenic alopecia is related to increased androgen metabolism in local hair follicles. Estrogen can inhibit the conversion of testosterone to dihydrotestosterone. Phytoestrogens can, to a certain extent, counteract androgens, increase local estrogen content, and alleviate hair loss. ISL has an estrogen receptor agonist effect. Estrogen can change the electrophysiological properties of neurons in a short period of time and reduce the Ca2+ level in neurons that do not express nuclear receptors. 2+ Current specifically enhances the activation of MAPK and other pathways, promoting cell proliferation.

[0033] Glycyrrhetinic acid (18β-Glycyrrhetinic acid, GA) is one of the main active components of licorice and a pentacyclic triterpenoid saponin of the oleanane type. It exhibits significant anti-inflammatory and antioxidant activities. Therefore, liposomes were constructed by replacing cholesterol with glycyrrhetinic acid to enhance the water solubility, hair follicle targeting, and stability of isoliquiritigenin. This resulted in the preparation of isoliquiritigenin-loaded glycyrrhetinic acid liposomes (GA-ISL-Lipo) for anti-AGA. Experimental results confirmed that the isoliquiritigenin-loaded glycyrrhetinic acid liposomes demonstrated significant hair coverage in the test animals on days 14 and 21 of topical administration. On day 21 of topical administration, hair length in the test animals was significantly longer than that in the AGA model animals. Topical application of these glycyrrhetinic acid liposomes also increased the number of subcutaneous hair follicles in the AGA model animals.

[0034] In a first aspect, a drug-loaded liposome is provided, which contains, in parts by weight, 20 to 50 parts by weight of lecithin, for example, but not limited to 20, 25, 30, 35, 40, 45 or 50 parts; 6 to 15 parts by weight of glycyrrhetinic acid, for example, but not limited to 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 parts; and 2 to 10 parts by weight of isoliquiritigenin, for example, but not limited to 2, 3, 4, 5, 6, 7, 8, 9 or 10 parts.

[0035] In an optional embodiment, the drug-loaded liposomes are prepared from raw materials comprising 30-50 parts by weight of lecithin, 9-15 parts by weight of glycyrrhetinic acid, and 5-10 parts by weight of isoliquiritigenin.

[0036] In an optional embodiment, the drug-loaded liposomes are prepared from raw materials comprising 50 parts by weight of lecithin, 15 parts by weight of glycyrrhetinic acid and 10 parts by weight of isoliquiritigenin.

[0037] In an optional embodiment, the drug-loaded liposomes are prepared from raw materials comprising 30 parts by weight of lecithin, 9 parts by weight of glycyrrhetinic acid and 5 parts by weight of isoliquiritigenin.

[0038] In an optional embodiment, the drug-loaded liposomes are prepared from raw materials comprising 50 parts by weight of lecithin, 15 parts by weight of glycyrrhetinic acid and 5 parts by weight of isoliquiritigenin.

[0039] In an optional embodiment, the drug-loaded liposome satisfies at least one of the following (i) to (iv): (i) the isoliquiritigenin loading in the liposomes is 5% to 20% w / w, for example, but not limited to, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20% w / w; (ii) the particle size of the liposome is 200-300 nm, for example, but not limited to, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290 or 300 nm; (iii) the liposomes have a PDI of 0.28 to 0.40; and, for example, but not limited to, 0.28, 0.285, 0.29, 0.295, 0.30, 0.31, 0.32, 0.33, 0.34, 0.35, 0.36, 0.37, 0.38, 0.39 or 0.40; (iv) The zeta potential of the liposome is -28 to -33 mV, for example, but not limited to, -28, -29, -30, -31, -32 or -33 mV.

[0040] In an optional embodiment, the drug loading amount of isoliquiritigenin in the drug-loaded liposomes is 5%~20% w / w, the particle size of the liposomes is 200~300 nm, the PDI is 0.28~0.40, and the zeta potential is -28~-33 mV.

[0041] In an optional embodiment, the drug-loaded liposomes are prepared by a method comprising the following steps: dissolving lecithin, glycyrrhetinic acid and isoliquiritigenin in an organic phase, rotary evaporating to form a dispersed film, injecting into an aqueous phase, incubating and then sonicating to obtain the drug-loaded liposomes.

[0042] In an optional embodiment, the organic phase includes 5000 to 10000 parts by weight of ethanol, and the ethanol may be, for example, but not limited to, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500 or 10000 parts by weight, preferably including 6000 to 8000 parts by weight of ethanol.

[0043] In an optional embodiment, lecithin, glycyrrhetinic acid and isoliquiritigenin are fully dissolved in the organic phase by ultrasound, and the ultrasound time is optionally 20 to 40 min, preferably 30 min.

[0044] In an optional embodiment, the rotary evaporation is carried out in a water bath at 40-60°C, preferably in a water bath at 50°C.

[0045] In an optional embodiment, the rotary evaporation time is 10 to 15 minutes.

[0046] In an optional embodiment, the incubation condition is 40-60° C. for 20-40 min, preferably 50° C. for 30 min.

[0047] In an optional embodiment, the incubation is followed by sonication for 5 to 15 minutes, preferably 10 minutes.

[0048] In a second aspect, a method for preparing drug-loaded liposomes loaded with isoliquiritigenin is also provided, the preparation method comprising dissolving lecithin, glycyrrhetinic acid and isoliquiritigenin in an organic phase, rotary evaporating to form a dispersed film, injecting into an aqueous phase, incubating and ultrasonicating to obtain the drug-loaded liposomes; wherein the weight proportion of lecithin is 20 to 50 weight proportions, for example, it can be but not limited to 20, 25, 30, 35, 40, 45 or 50 parts; the weight proportion of glycyrrhetinic acid is 6 to 15 weight proportions, for example, it can be but not limited to 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 parts; and the weight proportion of isoliquiritigenin is 2 to 10 weight proportions, for example, it can be but not limited to 2, 3, 4, 5, 6, 7, 8, 9 or 10 parts.

[0049] In an optional embodiment, in the preparation method, the weight parts of lecithin are 30 to 50 weight parts, the weight parts of glycyrrhetinic acid are 9 to 15 weight parts, and the weight parts of isoliquiritigenin are 5 to 10 weight parts.

[0050] In an optional embodiment, in the preparation method, the weight parts of lecithin is 50 weight parts, the weight parts of glycyrrhetinic acid is 15 weight parts, and the weight parts of isoliquiritigenin is 10 weight parts.

[0051] In an optional embodiment, in the preparation method, the weight parts of lecithin is 30 weight parts, the weight parts of glycyrrhetinic acid is 9 weight parts, and the weight parts of isoliquiritigenin is 5 weight parts.

[0052] In an optional embodiment, in the preparation method, the weight parts of lecithin is 50 weight parts, the weight parts of glycyrrhetinic acid is 15 weight parts, and the weight parts of isoliquiritigenin is 5 weight parts.

[0053] In an optional embodiment, the preparation method comprises the following steps: (a) dissolving lecithin, glycyrrhetinic acid, and isoliquiritigenin in 5,000 to 10,000 parts by weight of anhydrous ethanol, and sonicating to dissolve the lecithin, glycyrrhetinic acid, and isoliquiritigenin; (b) recovering anhydrous ethanol by rotary evaporation to form a dispersed film; (c) injecting pure water and incubating, and then sonicating after hydration to obtain the drug-loaded liposomes.

[0054] In an optional embodiment, in step (a), the weight parts of ethanol may be, for example, but not limited to, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500 or 10000 parts, preferably 6000-8000 parts by weight of ethanol.

[0055] In an optional embodiment, in step (a), the ultrasonic time is 20 to 40 min, preferably 30 min.

[0056] In an optional embodiment, in step (b), rotary evaporation is carried out in a water bath at 40-60°C, preferably in a water bath at 50°C.

[0057] In an optional embodiment, in step (b), the rotary evaporation time is 10 to 15 minutes.

[0058] In an optional embodiment, in step (c), the incubation condition is 40-60° C. for 20-40 min, preferably 50° C. for 30 min.

[0059] In an optional embodiment, in step (c), the ultrasonic time is 5 to 15 minutes, preferably 10 minutes.

[0060] In a third aspect, the invention further provides the use of the drug-loaded liposome described in the first aspect or the preparation method described in the second aspect in any of the following: (I) Preparation of medicaments for preventing and / or treating androgenic alopecia; (II) preparing a drug for alleviating inflammation; (III) Preparation of drugs for antioxidant activity; (IV) preparing a drug for inhibiting IL-6 and / or TNF-α; (V) Inhibition of IL-6 and / or TNF-α for non-diagnostic and non-therapeutic purposes.

[0061] In an optional embodiment, in the application, the drugs in (I) to (IV) are independently in the form of external dosage forms.

[0062] In an optional embodiment, in the use, IL-6 and / or TNF-α is inhibited by external application in (V).

[0063] In an optional embodiment, in the application, the inhibition of IL-6 and / or TNF-α in (V) includes inhibiting IL-6 and / or TNF-α in RAW264.7 macrophages.

[0064] In an alternative embodiment, the androgenic alopecia comprises testosterone-induced androgenic alopecia.

[0065] In an optional embodiment, the use for androgenic alopecia comprises at least one of increasing hair coverage, increasing hair length, and increasing the number of hair follicles.

[0066] In an optional embodiment, the increasing of hair length is increasing the length of new hair.

[0067] In an optional embodiment, the subject comprises a mouse animal model, preferably a testosterone-induced androgenic alopecia mouse model.

[0068] In a fourth aspect, a pharmaceutical composition is also provided, which contains the drug-loaded liposomes described in the first aspect, or the drug-loaded liposomes prepared by the preparation method described in the second aspect.

[0069] In an optional embodiment, the pharmaceutical composition is used for at least one of androgenic alopecia, relieving inflammation and anti-oxidation.

[0070] In an optional embodiment, the pharmaceutical composition is used to inhibit IL-6 and / or TNF-α.

[0071] In an optional embodiment, the pharmaceutical composition is in the form of an external dosage form.

[0072] In an optional embodiment, the pharmaceutical composition further comprises pharmaceutically optional excipients. Exemplary excipients include, but are not limited to, one or more of solvents, solubilizers, colorants, osmotic pressure regulators, stabilizers, antibacterial agents, suspending agents, antioxidants, and pH regulators.

[0073] The present invention is further described below by way of specific examples. However, it should be understood that these examples are merely provided for more detailed description and are not to be construed as limiting the present invention in any form.

[0074] Example 1: Preparation of Isoliquiritigenin Solution Isoliquiritigenin (1 g) was dissolved in preheated propylene glycol (2:1) and ultrasonically treated with heat until fully dissolved, yielding solutions with concentrations of 0.1%, 0.2%, and 0.4%. However, upon cooling, a large amount of solid isoliquiritigenin precipitated, rendering it unstable in the aqueous solution. Improving its solubility and stability was crucial for its application.

[0075] Example 2: Construction of liposomes loaded with isoliquiritigenin (1) Preparation of glycyrrhetinic acid liposomes loaded with isoliquiritigenin Lecithin, glycyrrhetinic acid and isoliquiritigenin were dissolved in anhydrous ethanol and ultrasonicated for 30 min to fully dissolve; in a 50°C water bath, the anhydrous ethanol was recovered by rotary evaporation for 10-15 min to form a dispersed film; 5 mL of pure water was injected, the mixture was incubated at 50°C for 30 min to fully hydrate, and ultrasonicated for 10 min to obtain glycyrrhetinic acid liposomes loaded with isoliquiritigenin.

[0076] (2) According to the preparation method of (1), glycyrrhetinic acid liposomes loaded with isoliquiritigenin were prepared in the following weight ratios of lecithin, glycyrrhetinic acid and isoliquiritigenin: 1) 30:9:5; 2) 30:9:10; 3) 50:15:5 and 4) 50:15:10. The weight ratio of anhydrous ethanol in 1) to 4) was 8000 parts.

[0077] (3) Determination of particle size potential The prepared drug liposomes were tested on a highly sensitive nanoparticle size analyzer Zetasizer Nano s90 to measure the particle size distribution of the glycyrrhetinic acid liposome sample loaded with isoliquiritigenin in pure water. The results are as follows: Figure 1 、 Figure 2 、 Figure 3 、 Figure 4 、 Figure 5 and Figure 6 The average particle size, dispersion index (PDI) and zeta potential were measured, and the results are shown in Table 1.

[0078] (4) Calculation of drug loading The prepared liposome solution was centrifuged at 15,000 rpm for 20 min, and the presence of precipitation was observed. The drug loading capacity was calculated according to the following formula.

[0079] .

[0080] After centrifugation, no precipitate was observed in the prepared liposomes, indicating that isoliquiritigenin could be completely encapsulated by the glycyrrhetinic acid liposomes.

[0081] Table 1 Average particle size, PDI and Zeta potential

[0082] The weight ratios in the above table are lecithin: glycyrrhetinic acid: isoliquiritigenin.

[0083] After comprehensive analysis of the particle size potential parameters of each liposome, liposomes with weight ratios of 30:9:5, 50:15:5, and 50:15:10 were selected, which had appropriate particle size, high uniformity and high stability, and were used for subsequent experiments.

[0084] Example 3: Comparison of the anti-inflammatory effects of liposomes containing glycyrrhetinic acid and cholesterol (1) Preparation of glycyrrhetinic acid liposomes and cholesterol liposomes As described in Example 2 (1), liposomes were prepared with phosphatidylcholine, glycyrrhetinic acid and isoliquiritigenin in weight ratios of 30:9:5, 50:15:5 and 50:15:10.

[0085] Liposomes having a weight ratio of lecithin, cholesterol and isoliquiritigenin of 30:9:5, 50:15:5 and 50:15:10 were prepared according to the method described in (1) under Example 2, except that glycyrrhetinic acid was replaced by an equal weight ratio of cholesterol.

[0086] (2) Cell culture RAW264.7 cells (mouse monocytes / macrophages) were subcultured in DMEM medium containing 10% fetal bovine serum, penicillin (100 U / mL), and streptomycin (100 U / mL) at 37°C and 5% CO2. All cells used in the experiments were in the logarithmic growth phase.

[0087] (3) Effects of glycyrrhetinic acid liposomes and cholesterol liposomes on the inflammatory level of RAW264.7 cells.

[0088] The cells were 1×10 6 100 μL of the solution was inoculated into each well of a 96-well plate and cultured in a 37°C, 5% CO2 constant temperature incubator. After the cells adhered to the wall, four groups were set up: The control group (containing an equal amount of solvent control), the model group (treated with 200 ng / mL LPS), the glycyrrhetinic acid liposomes loaded with isoliquiritigenin (LPS induction and drug loading concentrations of 5, 10, and 20 μmol / L, lecithin: glycyrrhetinic acid: isoliquiritigenin = 50:15:10), and the cholesterol-encapsulated isoliquiritigenin liposomes (LPS induction and drug loading concentrations of 5, 10, and 20 μmol / L, lecithin: cholesterol: isoliquiritigenin = 50:15:10) were used. After incubation for 12 h, the cell supernatants were collected and the IL-6 and TNF-α concentrations in the cell supernatants were determined using ELISA kits.

[0089] The results show that if Figure 7 and Figure 8 As shown in the results, glycyrrhetinic acid liposomes loaded with isoliquiritigenin had a better inhibitory effect on the LPS-induced increase in IL-6 and TNF-α levels.

[0090] Example 4: Comparison of the Antioxidant Effects of Liposomes Derived from Glycyrrhetinic Acid and Cholesterol (1) Preparation of DPPH (1,1-diphenyl-2-trinitrophenylhydrazine free radical) solution Accurately weigh DPPH and dilute it to volume in a 100 mL volumetric flask with methanol to obtain a 0.06 mmol / L DPPH ethanol solution.

[0091] (2) Sample addition and measurement Five sample concentrations were set: 10, 20, 50, 100, and 200 μmol / L. To 100 μL of a 0.1 mmol / L DPPH solution, 100 μL of the sample solution and anhydrous ethanol were added to bring the total volume to 200 μL. After incubation at room temperature in the dark for 30 minutes, 200 μL was transferred to a 96-well plate and mixed on a shaker for 10 seconds. The absorbance was measured at 517 nm, and the clearance rate was calculated.

[0092] ; Where: A0 is the absorbance of DPPH solution + anhydrous ethanol; A1 is the absorbance of DPPH solution + sample solution; A2 is the absorbance of sample solution + anhydrous ethanol.

[0093] The results show that Figure 9 As shown in the figure, glycyrrhetinic acid liposomes loaded with isoliquiritigenin have a more significant scavenging effect on DPPH free radicals than cholesterol liposomes with the same drug loading amount.

[0094] Example 5: Verification of the effectiveness of glycyrrhetinic acid liposomes loaded with isoliquiritigenin in preventing androgenic alopecia (1) Experimental methods and materials Six-week-old C57BL / 6J mice were purchased from the Experimental Animal Center of Southern Medical University. Twenty-four mice were randomly divided into a blank control group (NC), an androgenic alopecia group (AGA), an androgenic alopecia group treated with isoliquiritigenin-loaded glycyrrhetinic acid liposomes (AGA+ISL-Lipo), and an androgenic alopecia group treated with 2% minoxidil (AGA+MND), with six mice in each group. All mice except the blank control group were treated with an androgenic alopecia model according to the literature. After sodium pentobarbital anesthesia, the back hair of the mice was shaved with a razor, covering an area of ​​3 cm × 3 cm. Testosterone (testosterone (purity ≥95.0%) was slowly injected subcutaneously into the mice at a dose of 500 μL / mouse (5 mg / mL) in a solution of PEG-300, Tween-80, and saline). The blank control group received an equal volume of saline, which was administered subcutaneously for 28 consecutive days. Isoliquiritigenin-loaded glycyrrhetinic acid liposome solution was prepared and administered at a dose of 100 μL / mouse. The minoxidil group was given 2% minoxidil at a dose of 100 μL / mouse. The blank group and the androgenic alopecia group were given an equal amount of normal saline. The drugs were applied to the backs of the mice continuously for 21 days.

[0095] (2) Sampling of skin tissue from experimental animals After the last day of dosing and recording, skin tissue samples were collected. All instruments were prepared and disinfected before sampling. Each group of mice was anesthetized with an intraperitoneal injection of 50 mg / kg body weight of 1% sodium pentobarbital solution. After the mice were fully anesthetized, newly grown hair was shaved, and the dorsal skin of the mice was removed with tissue scissors, and the fascia was carefully removed. The separated skin was divided into two parts, one half was placed in a sterile 2 mL centrifuge tube and stored at -80°C; the other half was fixed in 4% paraformaldehyde at room temperature for 24 hours for subsequent tissue section staining.

[0096] (3) Recording of mouse hair growth 28 days after subcutaneous injection of testosterone, the mice were observed to see whether the hair follicles in the hair loss area on their backs were in the resting phase. After administration, photos were taken on days 0, 7, 14, and 21 to record the hair growth on the backs of the mice. Figure 10 As mentioned above, we observed the growth of hair on the backs of mice after continuous administration for 21 days. The results showed that the hair of mice with male pattern baldness grew slower than that of the blank control group, while the glycyrrhetinic acid liposomes loaded with isoliquiritigenin and minoxidil (positive control drug) significantly promoted the growth of hair on the backs of mice with male pattern baldness.

[0097] (4) Mouse hair coverage Image J software was used to calculate the hair coverage rate on the back of each mouse: Hair coverage rate (%) = hair growing area ÷ hair loss area × 100%. Figure 11As described above, we conducted a statistical analysis of hair coverage on days 7, 14, and 21. The results showed that on day 7, the early stage of drug administration, the hair coverage was significantly different only between the blank control group and the male pattern baldness model group, with the male pattern baldness model group having a significantly lower hair coverage than the blank control group (p < 0.01). On days 14 and 21, significant differences were found between the male pattern baldness model group and the blank control group, the glycyrrhetinic acid liposomes loaded with isoliquiritigenin, and the minoxidil-treated group (p < 0.01). Administration of isoliquiritigenin or minoxidil significantly increased the hair coverage of male pattern baldness model mice, revealing the potential role of glycyrrhetinic acid liposomes loaded with isoliquiritigenin in promoting hair growth in male pattern baldness model mice from a macroscopic perspective.

[0098] (5) Determination of mouse hair length On the 21st day of administration, 5 hairs were plucked from the front, middle and back of the depilatory area, and the hair length was measured using a vernier caliper. Figure 12 As mentioned above, we measured and analyzed the hair length of mice on day 21. The results showed that glycyrrhetinic acid liposomes loaded with isoliquiritigenin had the same effect as minoxidil, and both could significantly increase the hair length on the back of mice (p < 0.01).

[0099] Example 6: Effects of Isoliquiritigenin-Loaded Glycyrrhetinic Acid Liposomes on Hair Follicle Tissue After fixation, the tissues were dehydrated through a gradient of ethanol and xylene, paraffin-immersed at 65°C, embedded, and sectioned for hematoxylin-eosin staining. The staining procedure was as follows: 1) Paraffin dissolution: Sections were baked at 65°C for 30 minutes; 2) Dewaxing: After baking, sections were quickly immersed in xylene three times for 5 minutes each. 3) Gradient hydration: Place in 100%, 90%, and 80% ethanol for 2 minutes each, and then in distilled water for 2 minutes; 4) Nuclear staining: Stain with hematoxylin for 10 minutes, then rinse with running water to remove the hematoxylin; 5) Differentiation: Differentiate with 1% hydrochloric acid alcohol for 5 seconds, rinse with water, and soak in tap water to return to blue for 10 minutes; 6) Counterstaining: Use eosin stain for 3 minutes; 7) Gradient dehydration: Dehydrate with 80%, 90%, 95%, and 100% ethanol; 8) Transparent: Use dimethylformamide 3 times, 3 minutes each; 9) Sealing: Add a drop of neutral gum to the tissue and seal with a coverslip; 10) Observe and photograph under a microscope after 24 hours. Figure 13 As mentioned above, we performed HE staining on the skin of each group of mice. The results showed that the glycyrrhetinic acid liposomes loaded with isoliquiritigenin and the minoxidil group increased the number of skin hair follicles and slightly increased skin thickness compared with the male pattern baldness model group, but had no significant effect on the overall skin structure.

[0100] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the above embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. The drug-loaded liposome loaded with isoliquiritigenin is characterized in that: The drug-loaded liposome is prepared from raw materials comprising 20-50 parts by weight of lecithin, 6-15 parts by weight of glycyrrhetinic acid and 2-10 parts by weight of isoliquiritigenin.

2. The drug-loaded liposome according to claim 1, characterized in that The drug-loaded liposome is prepared from raw materials comprising 30-50 parts by weight of lecithin, 9-15 parts by weight of glycyrrhetinic acid and 5-10 parts by weight of isoliquiritigenin.

3. The drug-loaded liposome according to claim 2, characterized in that The drug-loaded liposomes are prepared from raw materials comprising 50 parts by weight of lecithin, 15 parts by weight of glycyrrhetinic acid, and 10 parts by weight of isoliquiritigenin; Or, prepared from raw materials comprising 30 parts by weight of lecithin, 9 parts by weight of glycyrrhetinic acid and 5 parts by weight of isoliquiritigenin; Alternatively, it is prepared from raw materials comprising 50 parts by weight of lecithin, 15 parts by weight of glycyrrhetinic acid and 5 parts by weight of isoliquiritigenin.

4. The drug-loaded liposome according to any one of claims 1 to 3, characterized in that The drug-loaded liposome satisfies at least one of the following (i) to (iv): (i) the isoliquiritigenin loading amount in the liposomes is 5% to 20% w / w; (ii) the particle size of the liposome is 200-300 nm; (iii) the PDI of the liposome is 0.28 to 0.40; and, (iv) The zeta potential of the liposome is -28 to -33 mV.

5. The drug-loaded liposome according to any one of claims 1 to 3, characterized in that The drug-loaded liposome is prepared by a method comprising the following steps: dissolving lecithin, glycyrrhetinic acid and isoliquiritigenin in an organic phase, rotary evaporating to form a dispersed film, injecting into an aqueous phase, incubating, and then ultrasonicating to obtain the drug-loaded liposome.

6. The drug-loaded liposome according to claim 5, characterized in that The organic phase includes 5000 to 10000 parts by weight of ethanol.

7. A method for preparing drug-loaded liposomes loaded with isoliquiritigenin, characterized in that: The method comprises dissolving lecithin, glycyrrhetinic acid and isoliquiritigenin in an organic phase, rotary evaporating to form a dispersed film, injecting into an aqueous phase, incubating and then ultrasonicating to obtain the drug-loaded liposome; The weight proportion of lecithin is 20 to 50 parts by weight, the weight proportion of glycyrrhetinic acid is 6 to 15 parts by weight, and the weight proportion of isoliquiritigenin is 2 to 10 parts by weight.

8. The preparation method according to claim 7, characterized in that The steps include: (a) dissolving lecithin, glycyrrhetinic acid, and isoliquiritigenin in 5,000 to 10,000 parts by weight of anhydrous ethanol, and sonicating to dissolve the lecithin, glycyrrhetinic acid, and isoliquiritigenin; (b) recovering anhydrous ethanol by rotary evaporation to form a dispersed film; (c) injecting pure water and incubating, and then sonicating after hydration to obtain the drug-loaded liposomes.

9. Use of the drug-loaded liposome according to any one of claims 1 to 6, or the preparation method according to claim 7 or 8, in any of the following: (I) Preparation of medicaments for preventing and / or treating androgenic alopecia; (II) preparing a drug for alleviating inflammation; (III) Preparation of drugs for antioxidant activity; (IV) preparing a drug for inhibiting IL-6 and / or TNF-α; (V) Inhibition of IL-6 and / or TNF-α for non-diagnostic and non-therapeutic purposes.

10. A pharmaceutical composition, characterized in that The invention comprises the drug-loaded liposome according to any one of claims 1 to 6, or the drug-loaded liposome prepared by the preparation method according to claim 7 or 8.

Images