Drug-loaded liposome of isoliquiritigenin, preparation method and application thereof

Abstract

The application provides a drug-loaded liposome loaded with isoglycyrrhizin, a preparation method and application thereof, and relates to the technical field of medicines.The drug-loaded liposome is prepared from raw materials containing lecithin 20-50 parts by weight, glycyrrhetic acid 6-15 parts by weight and isoglycyrrhizin 2-10 parts by weight, and can be used as a new drug for treating androgenetic alopecia.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of medicine, in particular to a drug-loaded liposome loaded with isoglycyrrhizin, a preparation method and application thereof. BACKGROUND

[0002] The following statements are only provided with the background information related to the present application, and not necessarily constitute the prior art.

[0003] Androgenetic alopecia (AGA) is the most common form of hair loss worldwide, caused by excessive androgen secretion, characterized by progressive terminal hair loss after puberty, accompanied by increased levels of inflammation and oxidative stress. The degree of androgenetic alopecia increases with age, and is distributed with unique characteristics of both sexes. Male pattern hair loss (MPHL) is characterized by gradual thinning of hair, mainly affecting the crown and forehead, and the hairline is receding; the characteristics of female pattern hair loss (FPHL) are diffuse hair loss and thinning, and the hairline is usually not affected.

[0004] Although the prevalence of AGA is high, the treatment of AGA can be challenging due to its chronic nature and the interaction of genetic and environmental factors. The current drugs for treating AGA mainly include topical minoxidil and oral finasteride. However, minoxidil has side effects such as headache, hirsutism, irritation and contact dermatitis; finasteride can cause sexual dysfunction, gynecomastia, mood changes and other adverse reactions.

[0005] Liposomes as drug carriers not only can promote the transdermal absorption of drugs, but also can maintain high concentration of drugs in the skin, and more importantly, liposomes have the characteristics of being close to hair follicles, which can achieve the directional enrichment of drug-loaded liposomes in hair follicles. Cholesterol is an important component of liposomes, which is inserted vertically to the lipid membrane bilayer plane, and the C-3 hydroxyl group forms a hydrogen bond with the polar head group of phospholipid, which has the function of maintaining the stability of the phospholipid bilayer membrane of liposomes. However, cholesterol is easily affected by oxygen, light and metal, and produces cholesterol oxidation products during processing and storage, which has certain cytotoxicity, mutagenicity and potential carcinogenicity.

[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 application is proposed. SUMMARY

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

[0009] In order to solve the above technical problems, the present application adopts the following technical solutions:

[0010] In a first aspect, a liposome loaded with glycyrrhetinic acid and isoliquirtin is provided, which is prepared from raw materials comprising lecithin 20-50 parts by weight, glycyrrhetinic acid 6-15 parts by weight and isoliquirtin 2-10 parts by weight.

[0011] In a second aspect, a preparation method of the liposome loaded with glycyrrhetinic acid and isoliquirtin is provided, which comprises dissolving lecithin, glycyrrhetinic acid and isoliquirtin in an organic phase, forming a dispersed film by rotary evaporation, injecting into an aqueous phase, incubating and then ultrasonicating to obtain the liposome.

[0012] In the preparation method, the lecithin is 20-50 parts by weight, the glycyrrhetinic acid is 6-15 parts by weight and the isoliquirtin is 2-10 parts by weight.

[0013] In a third aspect, the liposome of the first aspect or the preparation method of the second aspect is applied in any one of the following:

[0014] (I) preparation of a drug for preventing and / or treating androgenetic alopecia;

[0015] (II) preparation of a drug for relieving inflammation;

[0016] (III) preparation of a drug for resisting oxidation;

[0017] (IV) preparation of a drug for inhibiting IL-6 and / or TNF-α;

[0018] (V) inhibiting IL-6 and / or TNF-α for non-diagnostic and therapeutic purposes.

[0019] In a fourth aspect, a pharmaceutical composition is further provided, which comprises the liposome of the first aspect or the liposome prepared by the preparation method of the second aspect.

[0020] Compared with the prior art, the present application has the following beneficial effects:

[0021] The liposome provided by the application takes lecithin and glycyrrhetinic acid as a carrier, and loads isoliquiritigenin. Due to the characteristics of the liposome, the drug-loaded liposome has hair follicle targeting, and can be enriched in the hair follicle site. Isoliquiritigenin as a drug active ingredient can inhibit the conversion of testosterone into dihydrotestosterone by exerting its phytoestrogen characteristics, to a certain extent, against androgens, and then activate the hair follicle, so that the hair follicle changes from the resting phase to the growth phase, and promotes hair growth. In addition, the water solubility, hair follicle targeting and stability of isoliquiritigenin are enhanced by the liposome encapsulation technology, and the coverage area, hair length and hair follicle number of the hair of the androgenetic alopecia mice are promoted. The drug-loaded liposome provided by the application also has better anti-inflammatory and antioxidant effects. The application verifies the effect of the drug-loaded liposome loaded with isoliquiritigenin in the prevention and treatment of androgenetic alopecia, widens the application range of isoliquiritigenin, and provides a new candidate drug for the prevention and treatment of androgenetic alopecia. BRIEF DESCRIPTION OF DRAWINGS

[0022] In order to more clearly illustrate the technical solutions in the specific embodiments or prior art of the present application, the drawings needed in the specific embodiments or prior art description will be briefly introduced below. Obviously, the drawings in the following description are some embodiments of the present application, and other drawings can also be obtained by those skilled in the art without creative labor.

[0023] Figure 1 The particle size distribution diagram of the liposome sample with a weight ratio of lecithin, glycyrrhetinic acid and isoliquiritigenin of 20:15:2 in pure water in Example 2;

[0024] Figure 2 The particle size distribution diagram of the liposome sample with a weight ratio of lecithin, glycyrrhetinic acid and isoliquiritigenin of 30:9:5 in pure water in Example 2;

[0025] Figure 3 The particle size distribution diagram of the liposome sample with a weight ratio of lecithin, glycyrrhetinic acid and isoliquiritigenin of 30:9:10 in pure water in Example 2;

[0026] Figure 4 The particle size distribution diagram of the liposome sample with a weight ratio of lecithin, glycyrrhetinic acid and isoliquiritigenin of 50:15:5 in pure water in Example 2;

[0027] Figure 5 The particle size distribution diagram of the liposome sample with a weight ratio of lecithin, glycyrrhetinic acid and isoliquiritigenin of 50:15:10 in pure water in Example 2;

[0028] Figure 6 The particle size distribution diagram of the liposome sample with a weight ratio of lecithin, glycyrrhetinic acid and isoliquiritigenin of 50:6:10 in pure water in Example 2;

[0029] Figure 7 IL-6 concentration of each experimental group in Example 3, wherein NC is blank control group, LPS is LPS-induced inflammation group, GA-ISL-Lipo is glycyrrhetinic acid liposome loaded with isoliquirtin administration group, CHOL-ISL-Lipo is cholesterol liposome loaded with isoliquirtin administration group;

[0030] Figure 8 TNF-α concentration of each experimental group in Example 3, wherein NC is blank control group, LPS is LPS-induced inflammation group, GA-ISL-Lipo is glycyrrhetinic acid liposome loaded with isoliquirtin administration group, CHOL-ISL-Lipo is cholesterol liposome loaded with isoliquirtin administration group;

[0031] Figure 9 DPPH inhibition rate of GA-ISL-Lipo group and CHOL-ISL-Lipo group in Example 3, wherein GA-ISL-Lipo is glycyrrhetinic acid liposome loaded with isoliquirtin administration group, CHOL-ISL-Lipo is cholesterol liposome loaded with isoliquirtin administration group;

[0032] Figure 10 Figure of hair growth of testosterone-induced androgen alopecia mice promoted by glycyrrhetinic acid liposome loaded with isoliquirtin in Example 5, wherein NC is blank control group, AGA is testosterone-induced androgen alopecia group, AGA+GA-ISL-Lipo is androgen alopecia glycyrrhetinic acid liposome loaded with isoliquirtin treatment group, AGA+MND is androgen alopecia 2% minoxidil treatment group (positive control group); A is the back skin condition of mice after 28 days of subcutaneous injection of testosterone, the first day of administration, B is the back hair growth of mice on the 7th day of administration, C is the back hair growth of mice on the 14th day of administration, D is the back hair growth of mice on the 21st day of administration;

[0033] Figure 11 Figure of hair coverage of androgenetic alopecia mice promoted by glycyrrhetinic acid liposome loaded with isoliquirtin in Example 4, which is the hair coverage on the 7th, 14th and 21st day of administration, respectively, wherein NC is blank control group, AGA is testosterone-induced androgen alopecia group, AGA+GA-ISL-Lipo is androgen alopecia glycyrrhetinic acid liposome loaded with isoliquirtin treatment group, AGA+MND is androgen alopecia 2% minoxidil treatment group (positive control group);

[0034] Figure 12The graph of the length of the hair of the testosterone-induced androgen alopecia mice promoted by isofraxidin in Example 4, wherein NC is a blank control group, AGA is a testosterone-induced androgen alopecia group, AGA+GA-ISL-Lipo is an androgen alopecia treatment group loaded with isofraxidin glycyrrhetinic acid liposomes, and AGA+MND is a 2% minoxidil treatment group of androgen alopecia (positive control group).

[0035] Figure 13 The graph of the hematoxylin-eosin staining of the hair follicle tissue of the testosterone-induced androgen alopecia mice treated with isofraxidin in Example 6, wherein NC is a blank control group, AGA is a testosterone-induced androgen alopecia group, AGA+GA-ISL-Lipo is an androgen alopecia treatment group loaded with isofraxidin glycyrrhetinic acid liposomes, and AGA+MND is a 2% minoxidil treatment group of androgen alopecia (positive control group). DETAILED DESCRIPTION

[0036] The technical solutions of the present application will be described clearly and completely below in conjunction with the examples. Obviously, the described examples are only a part of the examples of the present application, rather than all the examples. Based on the examples in the present application, all the other examples obtained by a person of ordinary skill in the art without any creative effort fall within the protection scope of the present application.

[0037] In the present application, all the embodiments and preferred embodiments mentioned herein can be combined to form new technical solutions, if not specifically stated.

[0038] In the present application, all the technical features and preferred features mentioned herein can be combined to form new technical solutions, if not specifically stated.

[0039] In the present application, the components mentioned herein and the preferred components thereof can be combined to form new technical solutions, if not specifically stated.

[0040] In the present application, unless otherwise stated, the numerical range "a~b" represents a shorthand notation for any real number combination between a and b, wherein a and b are both real numbers. For example, the numerical range "6~22" represents that all the real numbers between "6~22" have been listed herein, and "6~22" is only a shorthand notation for these numerical combinations.

[0041] In the present application, the disclosed "range" in the form of lower limit and upper limit can be one or more lower limits and one or more upper limits, respectively.

[0042] In the present application, unless otherwise stated, each reaction or operation step can be performed in sequence or not in sequence. Preferably, the reaction method herein is performed in sequence.

[0043] As used herein, the term "inhibit," "reduce," "silence," "down-regulate," "repress," and other similar terms can be used interchangeably and include inhibition at any level. Inhibition can be assessed by a decrease 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 a subject, cell, or sample that has not been treated or has been treated with a control (e.g., a buffer control or an inert agent control).

[0044] As used herein, the terms "patient," "subject," or "individual" are used interchangeably and include a human or non-human animal, or a cell or tissue derived from a human or non-human animal, such as a human, monkey, mouse, rat, rabbit, donkey, cow, horse, pig, or dog.

[0045] As used herein, the terms "treat," "treatment," "alleviate," or "ameliorate" are used interchangeably herein and refer to methods of obtaining beneficial or desired results, including but not limited to therapeutic benefit. A "therapeutic benefit" means eradication or amelioration of one or more symptoms associated with the underlying disorder being treated.

[0046] As used herein, the terms "prevent," "prevention," "preventing," and "prevented" are used interchangeably herein and refer to methods of obtaining beneficial or desired results, including but not limited to prophylactic benefit. To obtain a "prophylactic benefit," a drug can be administered to a subject at risk of developing a particular disease, or to a subject reporting one or more of the physiological symptoms of a disease, even though a diagnosis of this disease can not have been made.

[0047] As used herein, the terms "comprise" or "comprising" mean including, but not limited to.

[0048] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. In addition, any method or material similar or equivalent to those described herein can be used in the practice of the present application.

[0049] In a first aspect, there is provided a drug-loaded liposome loaded with isoliquiritigenin, the drug-loaded liposome comprising lecithin, glycyrrhetinic acid and isoliquiritigenin.

[0050] Isoliquiritigenin (ISL) is a biomarker component in Glycyrrhiza uralensis Fisch, which has antioxidant and anti-inflammatory activities. The structure of isoliquiritigenin is shown in formula (I), which is an estrogen analogue.

[0051] Formula (I).

[0052] Glycyrrhiza uralensis Fisch is a traditional Chinese medicine with phytoestrogen activity, and ISL is one of the flavonoids with phytoestrogen activity in Glycyrrhiza uralensis Fisch. Androgenetic alopecia is associated with increased androgen metabolism in local hair follicles. Estrogen can inhibit the conversion of testosterone to dihydrotestosterone, and phytoestrogens can counteract androgens to some extent, increase local estrogen content, and reduce hair loss. ISL has estrogen receptor agonist effect. Estrogen can change the electrophysiological properties of neurons in a short time, reduce the Ca 2+ current of neurons that do not express nuclear receptors, and specifically enhance the activation of MAPK and other pathways to promote cell proliferation.

[0053] Glycyrrhetic acid (18β-Glycyrrhetinic acid, GA) is one of the main active components of Glycyrrhiza uralensis Fisch, belonging to oleanane pentacyclic triterpene saponins, with good anti-inflammatory and antioxidant activity. Therefore, by replacing cholesterol with glycyrrhetic acid to construct liposomes to enhance the water solubility, follicle targeting and stability of ISL, glycyrrhetic acid liposomes loaded with ISL (GA-ISL-Lipo) are prepared to resist AGA. The test proves that the glycyrrhetic acid liposomes loaded with ISL can make the test animals show obvious hair coverage on the 14th and 21st days of external administration; the hair length of the test animals is significantly longer than that of the AGA model animals on the 21st day of external administration; and the glycyrrhetic acid liposomes can promote the increase of the number of subcutaneous hair follicles in AGA model animals.

[0054] In a first aspect, a drug-loaded liposome is provided, which contains, in weight parts, lecithin 20-50 parts by weight, for example, but not limited to, 20, 25, 30, 35, 40, 45, or 50 parts; glycyrrhetic acid 6-15 parts by weight, for example, but not limited to, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 parts; and, ISL 2-10 parts by weight, for example, but not limited to, 2, 3, 4, 5, 6, 7, 8, 9, or 10 parts.

[0055] In an optional embodiment, the drug-loaded liposome is prepared from raw materials containing lecithin 30-50 parts by weight, glycyrrhetic acid 9-15 parts by weight, and ISL 5-10 parts by weight.

[0056] In an optional embodiment, the drug-loaded liposome is prepared from raw materials containing lecithin 50 parts by weight, glycyrrhetic acid 15 parts by weight, and ISL 10 parts by weight.

[0057] In an optional embodiment, the drug-loaded liposome is prepared from raw materials containing lecithin 30 parts by weight, glycyrrhetic acid 9 parts by weight, and ISL 5 parts by weight.

[0058] In an alternative embodiment, the drug-loaded liposome is prepared from raw materials comprising lecithin 50 parts by weight, glycyrrhetinic acid 15 parts by weight and isoliquiritigenin 5 parts by weight.

[0059] In an alternative embodiment, the drug-loaded liposome satisfies at least one of (i)-(iii) below:

[0060] (i) the drug loading amount of isoliquiritigenin in the liposome is 5%-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;

[0061] (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;

[0062] (iii) the PDI of the liposome is 0.28-0.40, 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;

[0063] (iv) the zeta potential of the liposome is -28--33 mV, for example, but not limited to, -28, -29, -30, -31, -32 or -33 mV.

[0064] In an alternative embodiment, the drug loading amount of isoliquiritigenin in the drug-loaded liposome is 5%-20% w / w, the particle size of the liposome is 200-300 nm, the PDI of the liposome is 0.28-0.40, and the zeta potential of the liposome is -28--33 mV.

[0065] In an alternative embodiment, the drug-loaded liposome is prepared by a method comprising the following steps: dissolving lecithin, glycyrrhetinic acid and isoliquiritigenin in an organic phase, rotary evaporation to form a dispersion film, injecting an aqueous phase, incubating and then ultrasonication to obtain the drug-loaded liposome.

[0066] In an alternative embodiment, the organic phase comprises 5000-10000 parts by weight of ethanol, for example, but not limited to, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500 or 10000 parts by weight, preferably 6000-8000 parts by weight of ethanol.

[0067] In an optional embodiment, the lecithin, glycyrrhetinic acid and glycyrrhizin are dissolved in the organic phase by ultrasonic for 20-40 min, preferably for 30 min.

[0068] In an optional embodiment, the rotary evaporation is performed in a water bath at 40-60 ℃, preferably in a water bath at 50 ℃.

[0069] In an optional embodiment, the rotary evaporation is performed for 10-15 min.

[0070] In an optional embodiment, the incubation is performed at 40-60 ℃ for 20-40 min, preferably at 50 ℃ for 30 min.

[0071] In an optional embodiment, the ultrasonic is performed for 5-15 min after the incubation, preferably for 10 min.

[0072] In a second aspect, a preparation method of the drug-loaded liposome loaded with glycyrrhizin is also provided, which comprises dissolving lecithin, glycyrrhetinic acid and glycyrrhizin in an organic phase, rotary evaporation to form a dispersed film, injection into an aqueous phase, incubation and ultrasonic to obtain the drug-loaded liposome; wherein the weight fraction of lecithin is 20-50 parts by weight, for example, but not limited to, 20, 25, 30, 35, 40, 45 or 50 parts; the weight fraction of glycyrrhetinic acid is 6-15 parts by weight, for example, but not limited to, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 parts; and the weight fraction of glycyrrhizin is 2-10 parts by weight, for example, but not limited to, 2, 3, 4, 5, 6, 7, 8, 9 or 10 parts.

[0073] In an optional embodiment of the preparation method, the weight fraction of lecithin is 30-50 parts by weight, the weight fraction of glycyrrhetinic acid is 9-15 parts by weight, and the weight fraction of glycyrrhizin is 5-10 parts by weight.

[0074] In an optional embodiment of the preparation method, the weight fraction of lecithin is 50 parts by weight, the weight fraction of glycyrrhetinic acid is 15 parts by weight, and the weight fraction of glycyrrhizin is 10 parts by weight.

[0075] In an optional embodiment of the preparation method, the weight fraction of lecithin is 30 parts by weight, the weight fraction of glycyrrhetinic acid is 9 parts by weight, and the weight fraction of glycyrrhizin is 5 parts by weight.

[0076] In an optional embodiment of the preparation method, the weight fraction of lecithin is 50 parts by weight, the weight fraction of glycyrrhetinic acid is 15 parts by weight, and the weight fraction of glycyrrhizin is 5 parts by weight.

[0077] In an optional embodiment, the preparation method comprises the following steps:

[0078] (a) dissolving lecithin, glycyrrhetinic acid and glycyrrhizin in 5000-10000 parts by weight of anhydrous ethanol, and ultrasonic dissolving lecithin, glycyrrhetinic acid and glycyrrhizin;

[0079] (b) recovering anhydrous ethanol by rotary evaporation to form a dispersed film;

[0080] (c) injecting pure water and incubating, and after hydration, ultrasonic treatment to obtain the drug-loaded liposome.

[0081] In an optional embodiment, in step (a), the parts by weight of ethanol may be, but are not limited to, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500 or 10000, preferably 6000-8000 parts by weight of ethanol.

[0082] In an optional embodiment, in step (a), the ultrasonic time is 20-40 min, preferably 30 min.

[0083] In an optional embodiment, in step (b), the rotary evaporation is performed in a water bath at 40-60℃, preferably in a water bath at 50℃.

[0084] In an optional embodiment, in step (b), the rotary evaporation time is 10-15 min.

[0085] In an optional embodiment, in step (c), the incubation conditions are 40-60℃ for 20-40 min, preferably 50℃ for 30 min.

[0086] In an optional embodiment, in step (c), the ultrasonic time is 5-15 min, preferably 10 min.

[0087] In a third aspect, the drug-loaded liposome of the first aspect or the preparation method of the second aspect is used in any one of the following:

[0088] (I) preparation of a medicament for preventing and / or treating androgenetic alopecia;

[0089] (II) preparation of a medicament for relieving inflammation;

[0090] (III) preparation of a medicament for antioxidation;

[0091] (IV) preparation of a medicament for inhibiting IL-6 and / or TNF-α;

[0092] (V) inhibiting IL-6 and / or TNF-α for non-diagnostic and therapeutic purposes.

[0093] In some embodiments, the application, the drug in (I)-(IV) is in a form suitable for topical administration.

[0094] In some embodiments, the application, the inhibiting IL-6 and / or TNF-α in (V) is by topical administration.

[0095] In some embodiments, the application, the inhibiting IL-6 and / or TNF-α in (V) comprises inhibiting IL-6 and / or TNF-α in RAW264.7 macrophages.

[0096] In some embodiments, the androgenic alopecia comprises testosterone-induced androgenic alopecia.

[0097] In some embodiments, the use in androgenic alopecia comprises at least one of increasing hair coverage, increasing hair length, and increasing the number of hair follicles.

[0098] In some embodiments, the increasing hair length comprises increasing anagen hair length.

[0099] In some embodiments, the subject comprises a mouse animal model, preferably a mouse animal model of testosterone-induced androgenic alopecia.

[0100] In a fourth aspect, there is provided a pharmaceutical composition comprising the drug-loaded liposome of the first aspect, or prepared by the method of the second aspect.

[0101] In some embodiments, the pharmaceutical composition is for at least one of androgenic alopecia, inflammation relief, and antioxidant.

[0102] In some embodiments, the pharmaceutical composition is for inhibiting IL-6 and / or TNF-α.

[0103] In some embodiments, the pharmaceutical composition is in a form suitable for topical administration.

[0104] In some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable excipient. Exemplary excipients include, but are not limited to, one or more of a solvent, a solubilizer, a colorant, an osmotic pressure adjusting agent, a stabilizer, a bacteriostatic agent, a suspending agent, an antioxidant, and a pH adjusting agent.

[0105] The present application is further described by the following specific examples, which should not be construed as limiting the application in any way.

[0106] Example 1: Preparation of Isoliquiritigenin Solution

[0107] Isoliquiritigenin 1 g was dissolved in preheated propylene glycol: water = 2:1, and heated with auxiliary ultrasonic treatment until completely dissolved, to make a solution with a concentration of 0.1%, 0.2%, 0.4% respectively. However, after standing and cooling, a large amount of isoliquiritigenin solid precipitated, which could not be stably maintained in the aqueous solution system, and its solubility and stability needed to be improved for use.

[0108] Example 2: Construction of Isoliquiritigenin-loaded Liposomes

[0109] (1) Preparation of Isoliquiritigenin-loaded Glycyrrhetic Acid Liposomes

[0110] Egg phospholipid, glycyrrhetic acid and isoliquiritigenin were dissolved in anhydrous ethanol, ultrasonicated for 30 min, and completely dissolved; water bath at 50°C, rotary evaporation for 10-15 min to recover anhydrous ethanol, forming a dispersed film; inject 5 mL of pure water, incubate at 50°C for 30 min, fully hydrate, ultrasonicate for 10 min, to obtain isoliquiritigenin-loaded glycyrrhetic acid liposomes.

[0111] (2) Isoliquiritigenin-loaded glycyrrhetic acid liposomes with the following weight ratio of egg phospholipid, glycyrrhetic acid and isoliquiritigenin were prepared according to the preparation method of (1): 1) 30:9:5; 2) 30:9:10; 3) 50:15:5 and 4) 50:15:10. The weight of anhydrous ethanol in 1) to 4) is 8000 parts.

[0112] (3) Particle size and potential determination

[0113] The prepared drug liposomes were detected by high-sensitivity nanoparticle size analyzer Zetasizer Nano s90 to determine the particle size distribution of isoliquiritigenin-loaded glycyrrhetic acid liposomes in pure water, and the results are shown in Figure 1 , Figure 2 , Figure 3 , Figure 4 , Figure 5 and Figure 6 , the average particle size, dispersion coefficient (PDI) and Zeta potential were determined, and the results are shown in Table 1.

[0114] (4) Drug loading calculation

[0115] The prepared liposome solution was centrifuged at 15000 rpm for 20 min, and the presence or absence of precipitate was observed, and the drug loading was calculated. The drug loading was calculated according to the following formula.

[0116] .

[0117] The prepared liposomes had no obvious precipitate after centrifugation, indicating that isoliquiritigenin could be completely encapsulated by glycyrrhetic acid liposomes.

[0118] Table 1 Average particle size, PDI and Zeta potential

[0119]

[0120] The weight ratio in the above table is lecithin: glycyrrhetic acid: glycyrrhizin.

[0121] The particle size and potential parameters of each liposome were comprehensively analyzed, and the liposomes with a weight ratio of 30:9:5, 50:15:5, and 50:15:10 were selected because they had appropriate particle size, high uniformity, and high stability, and were used for subsequent experiments.

[0122] Example 3: Comparison of the anti-inflammatory effects of liposomes of glycyrrhetic acid and cholesterol raw materials

[0123] (1) Preparation of glycyrrhetic acid liposomes and cholesterol liposomes

[0124] According to the method described in (1) of Example 2, liposomes with a weight ratio of 30:9:5, 50:15:5, and 50:15:10 of lecithin, glycyrrhetic acid, and glycyrrhizin were prepared.

[0125] Liposomes with a weight ratio of 30:9:5, 50:15:5, and 50:15:10 of lecithin, cholesterol, and glycyrrhizin were prepared according to the method described in (1) of Example 2, with the only difference being that glycyrrhetic acid was replaced with an equal weight of cholesterol.

[0126] (2) Cell culture

[0127] RAW264.7 cells (mouse monocyte / macrophage cells) 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. The cells used in the experiment were in the logarithmic growth phase.

[0128] (3) Effect of glycyrrhetic acid liposomes and cholesterol liposomes on the inflammatory level of RAW264.7 cells.

[0129] Cells were inoculated at 1×10 6 per well in a 96-well plate, and after the cells adhered to the wall in a 37°C, 5% CO2 incubator, four groups were set up:

[0130] The control group (containing the same amount of solvent control), the model group (LPS 200 ng / mL treatment), the glycyrrhetinic acid liposome loaded with isoliquirtin (LPS induction and 5, 10, 20 μmol / L drug loading concentration, lecithin: glycyrrhetinic acid: isoliquirtin = 50: 15: 10), the cholesteryl isoliquirtin liposome (LPS induction and 5, 10, 20 μmol / L drug loading concentration treatment, lecithin: cholesterol: isoliquirtin = 50: 15: 10), after 12 h of incubation, the cell supernatant was collected, and the IL-6 and TNF-α concentrations in the cell supernatant were determined according to the ELISA kit.

[0131] The results show that, as shown in Figure 7 and Figure 8 , the glycyrrhetinic acid liposome loaded with isoliquirtin has a better inhibitory effect on the increase of IL-6 and TNF-α levels induced by LPS.

[0132] Example 4: Comparison of the antioxidant effect of liposomes of glycyrrhetinic acid and cholesteryl raw materials

[0133] (1) Preparation of DPPH (1,1-diphenyl-2-trinitrobenzene hydrazine free radical) solution

[0134] Accurately weigh DPPH, and dilute in a 100 mL volumetric flask with methanol to obtain a 0.06 mmol / L DPPH ethanol solution.

[0135] (2) Sample addition and determination

[0136] Five sample concentrations were set, i.e. 10, 20, 50, 100, and 200 μmol / L; 100 μL of 0.1 mmol / L DPPH solution was added to 100 μL of sample solution and anhydrous ethanol to make the total volume reach 200 μL. After 30 min of room temperature and light avoidance, 200 μL was taken to a 96-well plate, mixed on a shaker for 10 s, and the absorbance was determined at 517 nm to calculate the clearance rate.

[0137] ;

[0138] In the formula: 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.

[0139] The results show that, as shown in Figure 9 , the glycyrrhetinic acid liposome loaded with isoliquirtin has a more significant DPPH free radical scavenging effect than the cholesteryl liposome with the same drug loading amount.

[0140] Example 5: Verification of the effectiveness of the glycyrrhetinic acid liposome loaded with isoliquirtin in anti-androgen alopecia

[0141] (1) Experimental methods and materials

[0142] The 6-week-old mice (C57BL / 6J) selected in this study were purchased from the Experimental Animal Center of Southern Medical University. Twenty-four mice were randomly divided into a blank control group (NC), an androgen alopecia group (AGA), an androgen alopecia isofraxidin liposome treatment group (AGA+ISL-Lipo), and an androgen alopecia 2% minoxidil treatment group (AGA+MND), with 6 mice in each group. Except for the blank control group, the remaining mice were subjected to androgen alopecia modeling according to the literature. After the mice were anesthetized with sodium pentobarbital, the hair on the back of the mice was removed with a hair clipper, and testosterone was slowly injected subcutaneously into the mice at a dose of 500 μL per mouse (testosterone (purity ≥ 95.0%) was dissolved in a PEG300, Tween 80, and normal saline system at a dose of 5 mg / mL). The blank control group was injected with an equal amount of normal saline, and the subcutaneous injection was performed continuously for 28 days. The isofraxidin liposome solution was prepared, and the drug dose was 100 μL per mouse. The minoxidil group was given 2% minoxidil at a dose of 100 μL per mouse. The blank group and the androgen alopecia group were given an equal amount of normal saline. The drug was continuously applied to the back of the mice for 21 days.

[0143] (2) Collection of skin tissue from experimental animals

[0144] After the last day of administration and recording, the skin tissue samples were collected. All instruments were prepared and sterilized before sampling. Each mouse was anesthetized by intraperitoneal injection of 50 mg / kg body weight of 1% sodium pentobarbital solution. After the mouse was completely anesthetized, the newly grown hair was removed, and the skin on the back of the mouse 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 placed in 4% paraformaldehyde at room temperature for 24 hours for subsequent tissue section staining.

[0145] (3) Recording of mouse hair growth

[0146] After the mice were subcutaneously injected with testosterone for 28 days, whether the hair loss area on the back of the mouse was in the hair follicle resting phase was observed. After administration, the hair growth on the back of the mouse was recorded by taking photos on days 0, 7, 14, and 21, respectively. As described above, we observed the hair growth on the back of the mouse after 21 days of continuous administration. The results showed that the hair growth of the androgen alopecia model mice was slower than that of the blank control group, while the isofraxidin liposome and minoxidil (positive control drug) administration groups could significantly promote the hair growth on the back of the androgen alopecia model mice. Figure 10

[0147] (4) Mouse hair coverage rate

[0148] ​ImageJ software was used to calculate the hair coverage rate on the back of each mouse: Hair coverage rate (%) = Area of ​​hair growth ÷ Area of ​​hair loss × 100%. Figure 11 We conducted statistical analysis on hair coverage on days 7, 14, and 21. The results showed that on day 7, the early stage of drug administration, there was a significant difference in hair coverage only between the blank control group and the androgenetic alopecia model group; the hair coverage in the androgenetic alopecia model group was significantly lower than that in the blank control group (p < 0.01). On days 14 and 21, there were significant differences between the androgenetic alopecia model group and the blank control group, the glycyrrhizin-loaded liposome group, and the minoxidil group (p < 0.01). Both isoglycyrrhizin and minoxidil administration significantly increased hair coverage in the androgenetic alopecia model mice, revealing the potential role of isoglycyrrhizin-loaded liposomes in promoting hair growth in androgenetic alopecia model mice from a macroscopic perspective.

[0149] (5) Measurement of mouse hair length

[0150] On day 21 of drug administration, five hairs were plucked from the front, middle, and back areas of the hair removal zone, and the hair length was measured using calipers. Figure 12 In this study, we measured and analyzed the hair length of mice on day 21. The results showed that glycyrrhizic acid liposomes loaded with isoglycyrrhizin had the same effect as minoxidil, both significantly increasing the hair length on the back of mice (p < 0.01).

[0151] Example 6: Effects of glycyrrhizin-loaded liposomes on hair follicle tissue

[0152] After fixation, the tissues were dehydrated by gradient ethanol and xylene, embedded in paraffin at 65°C, sectioned, and then stained with hematoxylin-eosin. The staining procedure was as follows: 1) Paraffin dissolution: The sections were baked at 65°C for 30 min; 2) Dewaxing: The baked sections were quickly immersed in xylene three times, for 5 min each time. 3) Gradient hydration: Soak in 100%, 90%, and 80% ethanol for 2 min each, then in distilled water for 2 min; 4) Nuclear staining: Stain with hematoxylin for 10 min, then rinse off the hematoxylin stain with running water; 5) Differentiation: Differentiate with 1% hydrochloric acid alcohol for 5 s, wash with water, and soak in tap water for 10 min to regain blue color; 6) Counterstaining: Stain with eosin for 3 min; 7) Gradient dehydration: Dehydrate with 80%, 90%, 95%, and 100% ethanol; 8) Clearing: Clear with dimethyl methacrylate three times, 3 min each time; 9) Mounting: Add one drop of neutral resin to the tissue and mount with a coverslip; 10) Observe and photograph under a microscope after 24 h. Figure 13According to the HE staining of the skin of each group of mice, the results showed that the glycyrrhetinic acid liposome loaded with isoliquiritigenin group and the minoxidil group increased the number of skin hair follicles compared with the castrated model group, slightly increased the skin thickness, but had no significant effect on the overall structure of the skin.

[0153] Finally, it should be noted that: the above examples are only used to illustrate the technical solutions of the present application, but not limited to them; although the present application has been described in detail with reference to the foregoing examples, those skilled in the art should understand that: it can still modify the technical solutions recorded in the foregoing examples, or make equivalent replacement for part or all of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present application.

Claims

1. Liposomes loaded with isoliquirtigenin, characterized in that, The drug-loaded liposome is prepared from raw materials of lecithin 30-50 parts by weight, glycyrrhetic acid 9-15 parts by weight and isoliquiritigenin 5-10 parts by weight.

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

3. The drug-loaded liposome according to claim 2, wherein, The drug-loaded liposome is prepared from raw materials of lecithin 50 parts by weight, glycyrrhetic acid 15 parts by weight and isoliquiritigenin 10 parts by weight. Or, prepared from raw materials of lecithin 30 parts by weight, glycyrrhetic acid 9 parts by weight and isoliquiritigenin 5 parts by weight. Or, prepared from raw materials of lecithin 50 parts by weight, glycyrrhetic acid 15 parts by weight and isoliquiritigenin 5 parts by weight.

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

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

6. The drug-loaded liposome according to claim 5, wherein, The organic phase comprises 5000-10000 parts by weight of ethanol.

7. A method for preparing a drug-loaded liposome loaded with isoliquirtigenin, characterized by, The drug-loaded liposome is prepared by a method comprising the following steps: dissolving lecithin, glycyrrhetic acid and isoliquiritigenin in an organic phase, rotary evaporation to form a dispersed film, injecting into an aqueous phase, incubating and then ultrasonic, to obtain the drug-loaded liposome. The drug-loaded liposome is prepared from raw materials of lecithin 30-50 parts by weight, glycyrrhetic acid 9-15 parts by weight and isoliquiritigenin 5-10 parts by weight.

8. The preparation method according to claim 7, characterized in that, The method comprises the following steps: (a) dissolving lecithin, glycyrrhetic acid and isoliquiritigenin in 5000-10000 parts by weight of anhydrous ethanol, and ultrasonic to dissolve lecithin, glycyrrhetic acid and isoliquiritigenin; (b) rotary evaporation to recover anhydrous ethanol to form a dispersed film; (c) injecting pure water and incubating, and ultrasonic after hydration to obtain the drug-loaded liposome.

9. Use of the drug-loaded liposome of any one of claims 1-6, or the drug-loaded liposome prepared by the preparation method of claim 7 or 8, in the preparation of a medicament for preventing and / or treating androgenic alopecia.

10. Use according to claim 9, characterized in that, The medicament for preventing and / or treating androgenic alopecia is also used for relieving inflammation, for antioxidant, for inhibiting IL-6, and / or for inhibiting TNF-α.

11. A pharmaceutical composition, characterized by, The drug-loaded liposome of any one of claims 1-6, or the drug-loaded liposome prepared by the preparation method of claim 7 or 8.

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