Method for preparing formononetin-encapsulated liposomes and cosmetic composition containing the same for alleviating or improving hair loss

Abstract

The present invention relates to a method for manufacturing a formononetin-capturing liposome and a cosmetic composition containing the same. The present invention provides a method for manufacturing a liposome capable of optimally capturing formononetin by exploring the optimal liposome components and their mixing ratios for the efficient capture of formononetin, and provides a cosmetic composition containing the liposome manufactured in this way.

Description

Technology Field

[0001] The present invention relates to a method for manufacturing a liposome that encapsulates formononetin and a cosmetic composition containing the same. More specifically, the invention aims to provide a method for manufacturing a liposome capable of optimally encapsulating formononetin by exploring the optimal liposome components and their mixing ratios for the efficient encapsulation of formononetin, and to provide a cosmetic composition for alleviating or improving hair loss containing the liposome manufactured in this way. Background Technology

[0003] The skin can be divided into the skin surface, epidermis, dermis, and subcutaneous tissue, and performs a barrier function to prevent intrusion from the outside. The stratum corneum on the skin surface is formed when keratinocytes flatten on the skin surface; 15 to 20 layers of keratinocytes bind tightly to each other to form a waterproof seal composed of lipids and proteins, thereby preventing useful substances from penetrating into the skin. Therefore, it is generally difficult to penetrate the stratum corneum in healthy skin, and even if it does, only very low concentrations pass through, which presents a problem in that bioactive substances contained in cosmetics cannot fully exhibit their functions.

[0004] Various studies are being conducted to address these skin penetration issues, with liposomes being a representative example. Liposomes possess both hydrophilic and hydrophobic regions, allowing them to incorporate both hydrophilic and lipid-soluble substances within their structure; utilizing this capability can improve the skin permeability of cosmetics.

[0005] Meanwhile, formononetin is a plant-based polyphenol of the isoflavone family and is a poorly soluble (fat-soluble) polyphenol. Therefore, without separate solubilization and capture action, it has the disadvantage of having very poor biological activity when applied to the skin.

[0006] Accordingly, the present invention sought to solve the problem of limited skin applicability of formononetin as described above through a liposome structure. Prior art literature

[0008] Korean Patent No. 10-2404209 (registration date May 26, 2022) describes a method for manufacturing liposomal vesicles using hydrogenated lechitin, cetyl alcohol, stearyl alcohol, phytosphingosine, and ceramide NP. The problem to be solved

[0009] In the present invention, as part of a method to improve the skin absorption or skin penetration of formononetin, we intend to develop and provide a technology utilizing liposomes. means of solving the problem

[0011] The present invention relates to a method for adding and mixing an oil phase to an aqueous phase, wherein the aqueous phase is purified water, and the oil phase is obtained by mixing hydrogenated lecithin, polyglyceryl-4 laurate, phytosphingosine, caprylic / capric triglyceride, and formononetin, then heating to dissolve and cooling to room temperature, and the composition of the purified water, hydrogenated lecithin, polyglyceryl-4 laurate, phytosphingosine, caprylic / capric triglyceride, and formononetin is the remainder of purified water; hydrogenated lecithin 1.50±10% by weight; polyglyceryl-4 laurate 3.00±10% by weight; phytosphingosine 0.01±10% by weight; and caprylic / capric triglyceride 0.01±10% by weight. A method for manufacturing a 'formononetin-capturing liposome' is provided, characterized by having 0.40±10% by weight of formononetin.

[0012] In the method for manufacturing a 'formononetin-capturing liposome' of the present invention, the 'formononetin-capturing liposome' may preferably have an improved skin absorption rate of formononetin.

[0013] The present invention provides a cosmetic composition containing a 'formononetin-capturing liposome' prepared by the method of the present invention.

[0014] In the cosmetic composition of the present invention, the cosmetic composition may preferably be for alleviating hair loss or promoting hair growth. Effects of the invention

[0016] The primary objective of the present invention was to improve the skin absorption or penetration of formononetin. In this invention, optimal liposome conditions suitable for the encapsulation of formononetin were established through physicochemical analysis, and the liposomes thus optimized and manufactured, along with polyphenols prior to encapsulation in vitro The efficacy was compared. As a result of the experiment, it was confirmed that liposomes encapsulating formononetin were highly effective even at a much lower concentration compared to general formononetin. In conclusion, it was confirmed that the 'formononetin-encapsulating liposomes' of the present invention are cosmetic ingredients that can help alleviate hair loss and promote hair growth. Brief explanation of the drawing

[0018] Figure 1 is a size distribution graph of several liposomes prepared in Example 1 of the present invention. Figure 2 shows the skin absorption rate of the optimal liposome (composition D liposome of Example 1) prepared in the present invention. Figure 3 is a electron microscope (TEM) image of the optimal liposome (composition D liposome of Example 1) prepared in the present invention. Figure 4 shows the results of testing the appropriate content of formononetin in HaCaT cells, a human keratinocyte cell line, as a preliminary experiment. Figure 5 shows the experimental results of the content of formononetin, which is effective on hair papilla cells. Figure 6 is the result of testing the content of 'formononetin-capturing liposomes (liposomes of composition D of Example 1)' which are effective on hair papilla cells. Specific details for implementing the invention

[0019] In this invention, we aimed to develop a cosmetic composition that helps promote the proliferation of hair papilla cells for the purpose of alleviating hair loss. To this end, we sought to help alleviate hair loss by utilizing formononetin among polyphenols. However, formononetin is a poorly soluble (fat-soluble) polyphenol, and it has the disadvantage of having very poor biological activity when applied without solubilization and capture action.

[0020] To solve this problem, the present invention utilized liposome capture technology. By capturing formononetin within liposomes, it was possible to process it into a material with high penetration and high physiological activity even in a small amount.

[0021] In the present invention, an optimization process was performed for the preparation of formononetin-capturing liposomes, and the physiological activity of uncaptured formononetin and liposome-capturing formononetin for dermal papilla cell proliferation was measured and compared. As a result of the experiment, it was confirmed that the formononetin-capturing liposomes in the present invention were prepared under optimized conditions, had good skin penetration, and exhibited increased physiological activity.

[0022] Accordingly, the present invention comprises adding and mixing an oil phase to an aqueous phase, wherein the aqueous phase is purified water, and the oil phase is obtained by mixing hydrogenated lecithin, polyglyceryl-4 laurate, phytosphingosine, caprylic / capric triglyceride, and formononetin, then heating to dissolve and cooling to room temperature, and the composition of the purified water, hydrogenated lecithin, polyglyceryl-4 laurate, phytosphingosine, caprylic / capric triglyceride, and formononetin is the remainder of purified water; hydrogenated lecithin 1.50±10% by weight; polyglyceryl-4 laurate 3.00±10% by weight; phytosphingosine 0.01±10% by weight; and caprylic / capric triglyceride 0.01±10% by weight. A method for manufacturing a 'formononetin-capturing liposome' is provided, characterized by having 0.40±10% by weight of formononetin.

[0023] In the present invention, hydrogenated lecithin, polyglyceryl-4 laurate, phytosphingosine, and caprylic / capric triglyceride were selected as optimal components for manufacturing liposomes capable of capturing formononetin.

[0024] Hydrogenated lecithin plays a key phospholipid role responsible for structural stability and formulation control in liposome manufacturing. Hydrogenated lecithin is a phospholipid with a hydrophilic head and a hydrophobic tail structure. In aqueous solution, it naturally forms a phospholipid bilayer to form the basic structure of the liposome and encapsulates the active ingredient (formononetin in the present invention) between the bilayers.

[0025] Polyglyceryl-4 Laurate (PG-4 Laurate) is a nonionic surfactant that acts as a co-surfactant and membrane property regulator in the preparation of liposomes according to the present invention. It promotes phospholipid dispersion and initial self-assembly, and prevents phospholipids from clumping in the aqueous phase, thereby reducing membrane formation energy.

[0026] Phytosphingosine plays a role in simultaneously enhancing membrane strength, biocompatibility, and functionality (skin / cell affinity) in the preparation of liposomes according to the present invention. In addition, it is inserted into the phospholipid bilayer of the liposome to perform a structural role.

[0027] Caprylic / Capric Triglyceride (CCT) plays a role in dissolving membrane-assisted lipids and lipid-soluble components in the preparation of liposomes according to the present invention. Caprylic / Capric Triglyceride acts as a dissolution matrix for lipid-soluble active ingredients, but formononetin, a lipid-soluble component, is not uniformly captured by phospholipids alone. At this time, caprylic / capric triglyceride acts as a pre-dissolution solvent to influence the homogenization of dispersion within the membrane, thereby increasing the capture efficiency.

[0028] In the present invention, formononetin is mixed with the above-mentioned hydrogenated lecithin, polyglyceryl-4 laurate, phytosphingosine, and caprylic / capric triglyceride, heated to dissolve the mixture, and then cooled to room temperature to prepare an oil phase. The oil phase thus prepared is added to an aqueous phase prepared with purified water and mixed, and liposomes are manufactured by self-assembly through mixing. In the manufacture of liposomes according to the present invention, additional technical details required for liposome manufacturing, other than those specifically described in the present invention, may be based on known technology, and accordingly, such specific description is omitted.

[0029] Meanwhile, in the present invention, the optimal ratio of components used to manufacture liposomes capable of effectively capturing formononetin was derived. In the present invention, the composition of purified water, hydrogenated lecithin, polyglyceryl-4 laurate, phytosphingosine, caprylic / capric triglyceride, and formononetin is preferably as follows: residual amount of purified water; hydrogenated lecithin 1.50±10% by weight; polyglyceryl-4 laurate 3.00±10% by weight; phytosphingosine 0.01±10% by weight; caprylic / capric triglyceride 0.01±10% by weight; and formononetin 0.40±10% by weight. In the above composition, '±10%' is a simplified expression indicating a range of '-10% to +10%' of the value listed above; for example, hydrogenated lecithin '1.50±10% by weight' means hydrogenated lecithin '1.35 to 1.65% by weight'.

[0030] In the composition of the present invention, the remainder being purified water; hydrogenated lecithin 1.50±10% by weight; polyglyceryl-4 laurate 3.00±10% by weight; phytosphingosine 0.01±10% by weight; caprylic / capric triglyceride 0.01±10% by weight; and formononetin 0.40±10% by weight, formononetin can be effectively captured, and as a result, skin absorption can be significantly improved.

[0032] Meanwhile, the present invention provides a cosmetic composition containing a 'formononetin-capturing liposome' prepared by the method of the present invention. The formulation of the cosmetic composition of the present invention may be prepared in a preferred form depending on the method of use, and examples may include a lotion, essence, hair lotion, hair cream, pack, foundation, hair shampoo, hair rinse, body wash, gel, spray, foam cleanser, ointment, etc.

[0033] In the cosmetic composition of the present invention, the cosmetic composition may preferably be for alleviating hair loss or promoting hair growth. The alleviation of hair loss or promotion of hair growth may result from enhancing or activating the growth of hair papilla cells.

[0035] The contents of the present invention will be explained in more detail below through the following examples and experimental examples. However, the scope of the present invention is not limited to the following examples and experimental examples, but includes variations of equivalent technical concepts.

[0037] [Example 1: Preparation of Formononetin-Containing Liposomes]

[0038] In this embodiment, five types (A to E) of formononetin-containing liposomes were prepared to identify the optimal liposome formulation for the encapsulation of formononetin. Hydrogenated lecithin used for liposome preparation is a component that forms the phospholipid bilayer of the liposome structure, providing a structure capable of encapsulating water-soluble components in the inner aqueous phase and lipid-soluble components in the outer oil phase. Additionally, it has polarity on its hydrophilic head, which plays a role in providing electrostatic stability to the formulation. If hydrogenated lecithin is excessive, the phospholipid bilayer becomes thicker or the particle size increases. Furthermore, as the particle size distribution widens and viscosity increases, liposomes become prone to collision or aggregation, which may lead to a decrease in the uniformity and dispersion stability of the formulation.

[0039] Accordingly, in Example 1, the focus was placed on controlling the concentration of hydrogenated lecithin to optimize the liposomes, and liposomes were prepared with the component ratios shown in Table 1 below. To prepare the liposomes, the oil phase was heated to dissolve and uniformly mixed, then cooled to room temperature. Subsequently, the mixture was homogenized using a homogenizer while the oil phase was added to the aqueous phase and mixed. Afterward, the mixture was degassed using a vacuum desiccator to remove air bubbles. Through this process, liposomes A to E were prepared. In addition to the formulation ratios listed in Table 1 below, other general technical details for liposome preparation were based on known technology.

[0040]

[0042] [Experimental Example 1: Physicochemical analysis of formononetin-containing liposomes prepared in Example 1 above]

[0043] In Experimental Example 1, physicochemical analysis was performed on several liposomes containing formononetin prepared in Example 1 above.

[0044] When the particle size is small in the nanoscale, the force of Brownian motion is dominant over gravity, resulting in colloidal stability with minimal sedimentation or creaming. Additionally, the uniform surface tension of the bilayer membrane allows for the uniform capture of active ingredients. Therefore, the small size and uniform particle size distribution of liposome particles serve as grounds for determining that the formulation is kinetically stable. The particle size of the liposomes was measured using a particle size analyzer (Zetasizer Advance Pro, Malvern Panalytical, UK). After diluting the liposomes 10-fold, 1 mL was collected, placed in a polystyrene cuvette, and measured using dynamic light scattering. The average data was taken after three measurements using the same method and is shown in Figure 1. It was determined that the smaller the particle size and the more uniform the particle distribution, the more stable the particles were. As shown in Figure 1, liposomes A, B, C, D, and E prepared in Example 1 exhibited the highest peaks at 17.66 nm, 46.68 nm, 11.23 nm, 9.65 nm, and 23.88 nm, respectively. In the case of E, a double peak rather than a single peak appeared, whereas in the case of D, a very narrow peak appeared at the smallest size, and as a result, it was determined that the particles were small and uniform.

[0045] Meanwhile, the potential at the shear plane of the electric double layer formed on the particle surface is called the zeta potential and is used as an indicator to evaluate the stability of dispersed colloids. When the zeta potential is low, the repulsive force between particles is low, leading to particle aggregation and an unstable dispersion state. However, if the absolute value of the zeta potential is 30 mV or higher, the electrical repulsive force is high, suppressing particle aggregation, and the dispersed phase can be judged to be electrostatically stable. The zeta potential of liposomes was measured using a particle size analyzer (Zetasizer Advance Pro, Malvern Panalytical, UK). The liposomes were diluted 10-fold and placed in a disposable folded capillary cell for measurement. After measuring three times using the same method, the average data of the results was taken and is shown in Table 2.

[0046]

[0047] The absolute zeta potential was 30 mV or higher in all liposome treatments, and was particularly pronounced in treatments D and E. In conclusion, D, which showed the smallest particle size and a relatively high zeta potential, was selected as the formononetin-loaded liposome composition, and the following tests were conducted.

[0049] [Experimental Example 2: Observation of skin absorption of optimal liposomes (liposomes with composition D of Example 1)]

[0050] In Experimental Example 2, the skin absorption rate was confirmed for the liposome composition D of Example 1, which was identified as the optimal liposome composition.

[0051] The skin absorption of the optimal liposome composition, 'Composition D Liposome,' was transmitted to an accredited testing institution to measure the skin absorption. The test was conducted on 20 male and female subjects aged 19 years or older. Liposomes were applied to the left forearm of the subjects in a space maintained under constant temperature and humidity conditions (22±2℃, 50±10%), and the skin absorption (absorption rate, speed, depth) efficacy before and after use was measured using 3D Ramna Spectroscopy (Tokyo Instruments, Inc., Japan).

[0052] The skin absorption rate of the optimal liposome (composition D liposome) was improved by 100.13% from 284128.60 AU to 568638.30 AU, the absorption rate was improved by 103.86% from 66.86 μm / h to 136.30 μm / h, and the skin absorption depth was improved by 103.86% from 33.43 μm to 68.15 μm (Fig. 2).

[0053] In conclusion, it was determined that the skin absorption rate of the optimal liposome (composition D liposome) was enhanced by more than 100%.

[0055] [Experimental Example 3: Electron Microscope (TEM) Image of Optimal Liposome (Composition D Liposome)]

[0056] In Experimental Example 3, electron microscope (TEM) images were taken of the optimal liposome (composition D liposome).

[0057] The result was as shown in Fig. 3. As seen in Fig. 3, the optimal liposome (composition D liposome) exhibited a uniform spherical shape of nano-size, similar to the particle size distribution.

[0059] [Experimental Example 4: Formononetin and Formononetin Optimized Capture Liposomes in vitro [Efficacy Comparison]

[0060] (1) Prior test of suitable formononetin content

[0061] In this experiment, we intended to measure the optimal efficacy of formononetin by first treating the human keratinocyte cell line HaCaT before applying it directly to dermal papilla cells.

[0062] Cell viability was measured using the MTT assay, and the method was as follows. Before treatment with the candidate substance, 1 x 10⁶ cells were placed in a 96-well plate. 3 The cells were resuspended in cells / well, and the medium was replaced every 2–3 days to promote cell growth. When the cells reached approximately 60% growth in each well, the material FM was applied at different concentrations; dermal papilla cells were cultured for 7 days, and human hair follicle cells for 2 days. After the reaction time was complete, MTT (0.5 mg / ml) was added to each well, and the cells were incubated at 37°C for 3 hours. For the formazan product, 200 µl of DMSO (dimethylsulfoxide) was added to each well and dissolved for 30 minutes, and the absorbance was measured at 565 nm using a Multifunction Microplate Reader.

[0063] The experimental results were as shown in Figure 4, and it was confirmed that when the concentration of formononetin was 400–800 ug / ml, it showed effective efficacy (growth enhancement or promotion).

[0065] (2) Treatment of actual dermal papilla cells with formononetin

[0066] Dermal papilla cells were proliferated as follows. Human dermal papilla cells (hDPC) were cultured in DMEM medium containing 10% FBS and 1% penicillin under conditions of 5% CO2 and 37°C. Since dermal papilla cells grow into normal cells at a very slow rate, the experiment was conducted to reflect this characteristic (e.g., when treating with each material, dermal papilla cells were treated for 7 days to confirm the cell growth effect).

[0067] Based on the concentrations confirmed above, when actually treated to dermal papilla cells, as shown in Figure 5, it was confirmed that there was an effect (growth enhancement or promotion) starting from a concentration of 200 ug / ml, and the effect was highest at 600 ug / ml.

[0069] (3) Results of treating dermal papilla cells with formononetin-optimized liposomes (composition D liposomes)

[0070] In this experiment, as in the above, we treated dermal papilla cells with formononetin-optimized capture liposomes (composition D liposomes).

[0071] The experimental results were shown in Figure 6, and the efficacy (growth enhancement or promotion) was exhibited starting from a concentration of 40 ug / ml, which is much smaller than the above '(2)', and it was confirmed that the efficacy was very good up to 60~100 ug / ml.

[0072] In conclusion, it was confirmed that liposomal formononetin showed much better efficacy (growth promotion or facilitation) than regular formononetin.

Claims

Claim 1 An oil phase is added to an aqueous phase and mixed, wherein the aqueous phase is purified water, and the oil phase is obtained by mixing hydrogenated lecithin, polyglyceryl-4 laurate, phytosphingosine, caprylic / capric triglyceride, and formononetin, then heating to dissolve and cooling to room temperature, and the composition of the purified water, hydrogenated lecithin, polyglyceryl-4 laurate, phytosphingosine, caprylic / capric triglyceride, and formononetin is the remainder of purified water; hydrogenated lecithin 1.50 wt%; polyglyceryl-4 laurate 3.00 wt%; phytosphingosine 0.01 wt%; caprylic / capric triglyceride 0.01 wt%; A method for manufacturing a 'formononetin-capturing liposome' characterized by containing 0.40% by weight of formononetin. Claim 2 A method for manufacturing a 'formononetin-capturing liposome' according to claim 1, wherein the 'formononetin-capturing liposome' is characterized by having an improved skin absorption rate of formononetin. Claim 3 A cosmetic composition containing a 'formononetin-capturing liposome' prepared by the method of claim 1. Claim 4 In paragraph 3, the cosmetic composition is characterized by being for alleviating hair loss.

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