Traditional Chinese medicine essential oil composition for treating alopecia and preparation method thereof

Abstract

The invention discloses a traditional Chinese medicine essential oil composition for treating alopecia, which comprises traditional Chinese medicine essential oil and a deep eutectic solvent, or the traditional Chinese medicine essential oil composition is a water-based traditional Chinese medicine essential oil transdermal delivery system prepared by dropwise adding the traditional Chinese medicine essential oil into the deep eutectic solvent, uniformly mixing and then uniformly mixing with water. The traditional Chinese medicine essential oil composition is simple in preparation process, can stably entrap essential oil, enhances percutaneous penetration of the essential oil through lipid extraction, effectively delivers the essential oil to hair follicles to play a role in promoting hair regeneration activity, has the effect of treating alopecia, especially androgen-derived alopecia, and has a good application prospect. The adverse reaction caused by applying medicines such as finasteride and minoxidil is avoided. The invention discloses application of the traditional Chinese medicine essential oil composition in preparation of a medicine with hair regeneration promoting activity, preferably application in preparation of a medicine for treating alopecia, and more preferably application in preparation of a medicine for treating androgen-derived alopecia.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical preparations and relates to a traditional Chinese medicine essential oil composition for treating hair loss and its preparation method, specifically a traditional Chinese medicine essential oil composition for treating androgenetic alopecia and its preparation method. Background Technology

[0002] Androgenetic alopecia (AGA), also known as seborrheic alopecia, is a condition characterized by a progressive decrease in hair density and is the most common type of hair loss in clinical practice. AGA can be divided into male pattern baldness and female pattern baldness based on the affected population, and they have different clinical manifestations. Early signs of male pattern baldness include a receding hairline in an "M" shape extending from the sides of the forehead to the crown, which, when connected to the bald patches on the top of the head, forms a characteristic "horseshoe" pattern. Female pattern baldness, on the other hand, is often characterized by diffuse thinning hair, typically manifesting as thinning hair on the crown, while the frontal hairline does not recede. AGA primarily affects adult men, and its prevalence is increasing annually, severely impacting the self-image, mental health, and quality of life of AGA patients.

[0003] The pathogenesis of alopecia areata (AGA) is very complex and not yet fully understood. Studies have shown that the pathogenic mechanism of AGA is mainly related to two androgens in the body: testosterone and dihydrotestosterone (DHT). Under the action of type II 5α-reductase in hair follicles, testosterone is converted to DHT, which then binds to androgen receptors to form androgen receptor complexes. These complexes enter the cell nucleus and act as transcription factors, inducing AGA. Therefore, effectively inhibiting the expression of type II 5α-reductase and androgen receptors is an effective strategy for treating AGA. Currently, the FDA-approved drugs for treating alopecia are only oral finasteride tablets and topical minoxidil, but both have serious adverse reactions. For example, oral finasteride can cause systemic adverse reactions such as decreased libido in men. It generally takes 4-9 months of continuous use to observe efficacy, and it is only effective for male AGA. Minoxidil typically requires continuous use for 3-6 months to observe any clinical effects, but it can cause adverse reactions such as allergic dermatitis and hirsutism. Skin irritation is mainly related to propylene glycol in the minoxidil solvent. Furthermore, a rebound effect can occur after discontinuing minoxidil, requiring indefinite use and significantly impacting patient compliance. Therefore, there is an urgent need to find a safe and effective drug to prevent and treat hair loss.

[0004] Essential oils (EOs) are volatile secondary metabolites derived from natural plants. They possess various pharmacological activities, including antibacterial, antioxidant, anti-inflammatory, anticancer, and antiviral properties, and are widely used in the pharmaceutical, food, and cosmetic industries. Due to their natural origin, essential oils have been widely used as an alternative therapy for androgenetic alopecia. Studies have shown that Platycladus orientalis leaf EOs have type II 5α-reductase inhibitory activity, which can promote hair regeneration. The antibacterial and anti-inflammatory activities of Platycladus orientalis leaf EOs can also improve micro-inflammation of hair follicles caused by excessive sebum secretion and excessive proliferation of scalp microorganisms due to dihydrotestosterone. EOs are complex, consisting of a mixture of dozens of bioactive substances, mainly containing lipid-soluble and highly volatile terpenes and oxygenated derivatives. Non-terpenes such as phenylpropionic acid give EOs their characteristic aromatic odor. These characteristics give EOs inherent properties such as high volatility, low solubility, instability to light and heat, oiliness, and a pungent odor. Currently, most arborvitae leaf essential oil preparations used to treat hair loss are compound essential oil preparations or transdermal delivery by dissolving the essential oil in organic solvents such as ethanol. Due to the highly concentrated nature of essential oils, direct topical application of compound essential oil preparations or the use of organic solvents can cause skin irritation and greasiness, severely affecting patient compliance. Furthermore, the high volatility and photothermal instability of essential oils mean that conventional preparations cannot guarantee the stability and efficacy of essential oils. Summary of the Invention

[0005] The purpose of this invention is to provide an easily prepared herbal essential oil composition. Based on deep eutectic solvent (DES) technology, DES is prepared using choline and geraniol, which have good biocompatibility and high safety. Complex components in essential oils (EO) are adsorbed through the hydrogen bonding network within the DES. Under π-π stacking and hydrophobic interactions, an aqueous transdermal delivery system (AqED) for herbal essential oils is assembled in an aqueous solution. This delivery system can stably encapsulate EO, enhance EO transdermal penetration, and exhibit significant hair regeneration-promoting activity.

[0006] The objective of this invention is achieved through the following technical solution:

[0007] A traditional Chinese medicine essential oil composition for treating hair loss, comprising a traditional Chinese medicine essential oil and a deep eutectic solvent.

[0008] The volume ratio of the herbal essential oil to the deep eutectic solvent is 1:1 to 1:80.

[0009] Preferably, the volume ratio of the herbal essential oil to the deep eutectic solvent is 1:1.6 to 1:62.

[0010] More preferably, the volume ratio of the herbal essential oil to the deep eutectic solvent is (0.5-2):(31-33).

[0011] More preferably, the volume ratio of the herbal essential oil to the deep eutectic solvent is (1-2):(32-33).

[0012] Specifically, the volume ratio of the herbal essential oil to the deep eutectic solvent is 0.5:31, 1:32, 2:33, 1:12.3, 1:7.6, 1:4.7, 1:2.5, 1:1.67, 1:3.5, 1:16.7, or 1:49.

[0013] The herbal essential oil composition for treating hair loss is obtained by adding herbal essential oil dropwise into a deep eutectic solvent and mixing them evenly.

[0014] The aforementioned herbal essential oil composition for treating hair loss is a transdermal delivery system of herbal essential oils and deep eutectic solvents, which are mainly self-assembled and combined through π-π stacking interactions and hydrophobic interactions.

[0015] To improve the oiliness of essential oils (EO), mask their irritating odor, enhance patient compliance, avoid skin irritation from EO and DES, and improve the stability of the delivery system, a preferred technical solution for a traditional Chinese medicine essential oil composition for treating hair loss according to the present invention is prepared by adding essential oil dropwise to a deep eutectic solvent, mixing thoroughly, and then mixing thoroughly with water to obtain an aqueous transdermal delivery system for traditional Chinese medicine essential oils.

[0016] In the aforementioned transdermal delivery system for traditional Chinese medicine essential oils, the volume percentage of the traditional Chinese medicine essential oils is 0.5-30%, the volume percentage of the deep eutectic solvent is 29.5-70%, and the remainder is water.

[0017] Preferably, in the transdermal delivery system for traditional Chinese medicine essential oils, the volume percentage of the traditional Chinese medicine essential oil is 0.5-2%, the volume percentage of the deep eutectic solvent is 31-33%, and the remainder is water.

[0018] More preferably, in the transdermal delivery system for traditional Chinese medicine essential oils, the volume percentage of the traditional Chinese medicine essential oil is 1-2%, the volume percentage of the eutectic solvent is 32-33%, and the remainder is water.

[0019] Specifically, in the aforementioned transdermal delivery system for traditional Chinese medicine essential oils, the volume percentage of the traditional Chinese medicine essential oil is 0.5%, the volume percentage of the deep eutectic solvent is 31%, and the remainder is water; or the volume percentage of the traditional Chinese medicine essential oil is 1%, the volume percentage of the deep eutectic solvent is 32%, and the remainder is water; or the volume percentage of the essential oil is 2%, the volume percentage of the deep eutectic solvent is 33%, and the remainder is water.

[0020] The herbal essential oil is selected from at least one of common essential oils such as arborvitae leaf essential oil and rosemary essential oil, with arborvitae leaf essential oil being preferred.

[0021] The aforementioned arborvitae leaf essential oil is a fraction obtained by steam distillation of fresh arborvitae leaves at a temperature of 40–60°C.

[0022] Specifically, the arborvitae leaf essential oil is prepared by the following method: take fresh arborvitae leaves, chop them, put them into an extraction tank, turn on the cooling water, then turn on the direct steam, control the pressure in the extraction tank at 0-0.15MPa, the distillation rate at about 6-200L / h, distill and extract for 3 hours, and collect the distillate at a temperature of 40-60℃, which is the arborvitae leaf essential oil.

[0023] The arborvitae leaf essential oil contains (+)-α-pinene, cis-linalool-4(15),5-diene, juniperene, D-limonene, trans-acorene, ronaene, β-caryophyllene, juniper-3,5-diene, geraniol, α-penetrone, elemol, myrcene, 1-Ethynyl-8-propyltricyclo[4.4.0.0(3,8)]decane, borneol acetate, (+)-4-carene, (-)-4-terpineol, β-malene, α-ionol, (+)-epibresinol, acetylphenol, taurolol, etc.

[0024] Furthermore, the arborvitae leaf essential oil contains (+)-α-pinene, sapinene, myrcene, (+)-3-carene, D-limonene, γ-terpinene, isoterpinene, (-)-4-terpineol, borneol acetate, (+)-4-carene, α-ionol, β-elemene, β-caryophyllene, (-)-thujone, juniper-3,5-diene, cis-ilanilide-4(15),5-diene, geraniol, ronaene, 4,9-Bauhinia diene, trans-acorene, elemenol, 1,2, 3,4-Tetrahydro-6-methyl-1-methylene-4-isopropylnaphthalene, (+)-epibentenol, β-marinolene, 1-Ethynyl-8-propyltricyclo[4.4.0.0(3,8)]decane, α-penetrone, cis-7-Isopropyl-10-methyl-4-oxo-bicyclodec-5-ene, dehydrorosinane, acetylenol, (+)-ferricylmethyl ether, taurin, etc.

[0025] The deep eutectic solvent is a low eutectic mixture formed by hydrogen bond donors and hydrogen bond acceptors, and the melting point of the deep eutectic solvent is typically below 100°C.

[0026] The molar ratio of the hydrogen bond acceptor to the hydrogen bond donor is 1:2.

[0027] The hydrogen bond donor is selected from at least one of organic acids, polyols, and sugars; the hydrogen bond acceptor is selected from at least one of quaternary ammonium salts and zwitterionic surfactants.

[0028] Preferably, the hydrogen bond donor is geraniol, and the hydrogen bond acceptor is choline or its bicarbonate.

[0029] The deep eutectic solvent is prepared by the following method: using water as the reaction solvent, an aqueous solution of hydrogen bond acceptor is placed in a water bath, and under stirring conditions, an aqueous solution of hydrogen bond donor is added dropwise to the aqueous solution of hydrogen bond acceptor until no carbon dioxide is released; after the reaction is completed, the solvent in the reaction solution is removed by vacuum evaporation, and then the product is dried in a vacuum drying oven to obtain the deep eutectic solvent.

[0030] The temperature of the water bath is 35-40℃, preferably 40℃; the reaction time is 12h.

[0031] The temperature of the vacuum evaporation is 50-60℃, preferably 60℃, and the time of the vacuum evaporation is 2 hours.

[0032] The drying temperature is 50-60℃, preferably 60℃, and the drying time is 24-48h, preferably 24h.

[0033] A method for preparing the aforementioned Chinese herbal essential oil composition includes: adding the Chinese herbal essential oil dropwise into a deep eutectic solvent, vortexing and mixing evenly, and then mixing evenly with water to prepare an aqueous Chinese herbal essential oil transdermal delivery system.

[0034] Specifically, the essential oil of traditional Chinese medicine is added dropwise to a deep eutectic solvent and vortexed at 2800 r / min and room temperature until uniform. Then, an appropriate amount of water is added and vortexed at room temperature and 2800 r / min until clear, thus preparing an aqueous transdermal delivery system of traditional Chinese medicine essential oil.

[0035] Another object of the present invention is the use of the aforementioned herbal essential oil composition in the preparation of a medicine with hair regeneration-promoting activity, preferably in the preparation of a medicine for treating hair loss, and more preferably in the preparation of a medicine for treating androgenetic alopecia.

[0036] The drug is a topical solution preparation.

[0037] The beneficial effects of this invention are:

[0038] The preparation process of the herbal essential oil composition of this invention is simple, which can stably encapsulate the essential oil and enhance the transdermal penetration of the essential oil through lipid extraction, effectively delivering the essential oil to the hair follicle to exert hair regeneration activity. It has the effect of treating hair loss, especially androgenetic alopecia, and avoids the adverse reactions caused by the use of drugs such as finasteride and minoxidil. Attached Figure Description

[0039] Figure 1This is a schematic flowchart illustrating the preparation of the herbal essential oil composition of the present invention.

[0040] Figure 2 This is the total ion chromatogram of the Platycladus orientalis leaf essential oil in the herbal essential oil composition of the present invention.

[0041] Figure 3 This is the Fourier transform infrared spectrum of the eutectic solvent in the herbal essential oil composition of the present invention.

[0042] Figure 4 The 1H NMR spectrum of the eutectic solvent in the herbal essential oil composition of this invention; wherein... Figure 4 A is the 1H NMR spectrum of choline, geranilic acid and deep eutectic solvent; Figure 4 B is the 1H NMR spectrum of the eutectic solvent.

[0043] Figure 5 Thermal analysis of the deep eutectic solvent in the herbal essential oil composition of this invention; wherein, Figure 5 A represents the differential scanning calorimetry curve of the eutectic solvent; Figure 5 B is the thermogravimetric analysis spectrum of the eutectic solvent.

[0044] Figure 6 This is a ternary phase diagram of the herbal essential oil composition of the present invention.

[0045] Figure 7 The figure shows the experimental results of the homogeneity of the herbal essential oil composition of the present invention.

[0046] Figure 8 The figure shows the results of the centrifugal stability test of the herbal essential oil composition of the present invention.

[0047] Figure 9 This is a diagram showing the dynamic light scattering experimental results of the herbal essential oil composition of this invention.

[0048] Figure 10 This is a transmission electron microscope image of the herbal essential oil composition of the present invention.

[0049] Figure 11 This is a small-angle X-ray scattering (SAXS) result of the herbal essential oil composition of this invention.

[0050] Figure 12 Characterization of the essential oil-eutectic solvent in the herbal essential oil composition of this invention; wherein, Figure 12 A represents the Fourier transform infrared spectra of essential oil, deep eutectic solvent, and essential oil-deep eutectic solvent; Figure 12 B represents the hydrogen nuclear magnetic resonance spectra of essential oil, eutectic solvent, and essential oil-eutectic solvent.

[0051] Figure 13 This is the ultraviolet-visible absorption spectrum of the herbal essential oil composition of the present invention; wherein, Figure 13A is the UV-Vis absorption spectrum of choline and geranilic acid in anhydrous ethanol in 1% AqED, deep eutectic solvent; Figure 13 B represents the UV-Vis absorption spectra of 1% AqED in water and DMSO, respectively. Figure 13 C represents the UV-Vis absorption spectrum of 1% AqED in 0.2% (w / v) SDS; Figure 13 D is the UV-Vis absorption spectrum of 1% AqED in the presence of gradient concentrations (0, 2, 4, 6 M) of urea; Figure 13 UV-Vis absorption spectra of 1% AqED in the presence of NaCl at gradient concentrations (0, 0.25, 0.5, 1M).

[0052] Figure 14 This is a diagram showing the results of an in vitro skin penetration experiment of the herbal essential oil composition of this invention; wherein, Figure 14 A is the standard curve of fluorescence intensity versus concentration of FITC fluorescent dye; Figure 14 B represents the in vitro skin permeation curve of the herbal essential oil composition.

[0053] Figure 15 This is a diagram showing the in vivo skin penetration test results of the herbal essential oil composition of this invention; wherein, Figure 15 A represents the in vivo transdermal fluorescence observation of the traditional Chinese medicine essential oil composition; Figure 15 B represents a semi-quantitative analysis of fluorescence intensity.

[0054] Figure 16 This is a diagram showing the experimental results of the transdermal mechanism of the herbal essential oil composition of this invention; wherein, Figure 16 A represents a microscopic observation of the stratum corneum of the skin; Figure 16 B represents the ATR FITR scan spectrum of the stratum corneum before and after treatment with the formulation.

[0055] Figure 17 This is a photograph of the skin on the back of a mouse taken during the hair regeneration activity experiment of this invention.

[0056] Figure 18 This represents the coverage rate of newly grown hair on day 14 in the hair regeneration activity experiment of this invention.

[0057] Figure 19 The figure shows the experimental results of the safety evaluation of the herbal essential oil composition of the present invention. Detailed Implementation

[0058] All raw materials or reagents used in this invention are commercially available.

[0059] The technical solution of the present invention will be further described below with reference to specific implementation examples.

[0060] Example 1: Extraction and Chemical Composition Analysis of Platycladus orientalis Leaf Essential Oil

[0061] Extraction process:

[0062] Take 300g of fresh arborvitae leaves, chop them, put them into the extraction tank, turn on the condenser, then turn on the direct steam, control the pressure in the extraction tank at 0-0.15MPa, the distillation rate at about 6-200L / h, distill for 3 hours, collect the distillate at 40-60℃, which is the arborvitae leaf essential oil (EO), and store the arborvitae leaf essential oil in a brown bottle.

[0063] Chemical composition analysis:

[0064] Gas chromatography conditions: The column was an HP-5MS quartz capillary column (30m × 250μm × 0.25μm). The initial temperature was 80℃ for 4 min, then increased to 160℃ at a rate of 6℃ / min and held for 5 min. The temperature was then increased to 170℃ at a rate of 2℃ / min and held for 4 min. Finally, the temperature was increased to 270℃ at a rate of 5℃ / min for a total duration of 35 min. High-purity nitrogen was used as the carrier gas at a flow rate of 1 mL / min.

[0065] Mass spectrometry conditions: EI ion source, electron energy 70 eV; electron multiplier voltage: 1.4 kV; ion source temperature: 230 °C; interface temperature: 200 °C; injection port temperature: 280 °C; column inlet pressure: 47 kPa; scan mode: Scan; mass scan range: 33-500 amu.

[0066] Sample preparation: Take 10 μL of Platycladus orientalis leaf EO, dilute with ethyl acetate to 1 μL / mL, filter through a 0.22 μm microporous membrane, and then inject for analysis. The injection volume is 1 μL, and the split ratio is 5:1.

[0067] Total ion current chromatogram of EO from Platycladus orientalis leaves as shown in the figure. Figure 2 As shown, about 31 chemical components were isolated. Table 1 shows the chemical components of Platycladus orientalis leaf EO. The results show that the chemical components with relatively high content in Platycladus orientalis leaf EO include α-pinene, cis-ethanoyl-4(15),5-diene, trans-acorene, juniperene, and rounaene.

[0068] Table 1: Chemical composition of EO from Platycladus orientalis leaves

[0069]

[0070]

[0071] Example 2: Preparation and Characterization of DES

[0072] A choline (Ch) bicarbonate aqueous solution (80% by mass, 0.01 mol of choline bicarbonate) was placed in a 100 mL round-bottom flask and heated in a 40 °C water bath. Geranilic acid (Ge) aqueous solution (85% by mass, 0.02 mol of geranilic acid) was added dropwise under stirring at 200 r / min. The reaction was allowed to proceed for 12 h, at which point no carbon dioxide was observed to be released. The round-bottom flask was then transferred to a rotary evaporator and evaporated at 60 °C for 2 h to remove the solvent. The product was then dried in a vacuum drying oven at 60 °C for 24 h to obtain DES.

[0073] The physicochemical properties of DES, including appearance, density, viscosity, and conductivity, were characterized. DES was prepared by a one-step salt metathesis reaction of Ch and Ge. It is a yellow, transparent, viscous liquid with certain fluidity and characteristic odor at room temperature. The density of DES is 0.97±0.01 g / mL, the viscosity is 646±45 mPa·s, the pH range is 6-7, and the conductivity is 11.56±0.65 mS / m.

[0074] Fourier transform infrared spectroscopy (FTIR) and nuclear magnetic resonance spectroscopy were used. 1 H-NMR was used to verify whether DES was successfully prepared. Figure 3 and Figure 4 The images show the Fourier transform infrared spectrum and the hydrogen nuclear magnetic resonance spectrum of the eutectic solvent in the herbal essential oil composition of this invention. Figure 3 As shown, compared to Ge, the polymerization hydroxyl stretching vibration peak in DES decreased from 3421.7 cm⁻¹. -1 Displaced to 3365.4cm -1 The stretching vibration peak of the carbonyl group is from 1691.7 cm⁻¹. -1 Displaced to 1692.4cm -1 This indicates the presence of strong hydrogen bonding interactions in DES. For example... Figure 4 As shown in Figure A, compared to Ch and Ge, the carboxyl hydrogen in Ge and the hydroxyl hydrogen in Ch disappear in DES. The formation of hydrogen bonds and the high viscosity of the DES system increase the relaxation rate of hydrogen-bonded protons, which may be the reason for the signal disappearance. The strong hydrogen bond interaction between Ge and Ch allows for rapid exchange of protons between the carboxyl group of Ge and the hydroxyl group of Ch, resulting in the neutralization of acidic protons in Ge. Figure 4 B). This demonstrates that the present invention successfully prepared DES.

[0075] Differential scanning calorimetry (DSC) and thermogravimetric analysis (TGA) were used to perform thermal analysis on DES. The DSC spectrum is shown below. Figure 5 As shown in Figure A, the glass transition temperature (T) g The temperature was -75.91℃, indicating that the liquid DES solidified into an amorphous solid. The TGA spectrum is shown below. Figure 5As shown in Figure B, DES lost approximately 4% of its weight at 155.48℃, likely due to the evaporation of moisture. It began to decompose at around 181℃. (Decomposition temperature T...) dec The temperature was 216.37℃.

[0076] Example 3: Preparation and Characterization of AqED

[0077] A suitable amount of DES (prepared in Example 2) was added, along with a certain amount of Platycladus orientalis leaf EO (prepared in Example 1). The mixture was vortexed at room temperature and a rotation speed of 2800 r / min. A suitable amount of water was then added, and the mixture was vortexed again at room temperature and a rotation speed of 2800 r / min until clear, thus preparing an aqueous EO-DES system (AqED). The volume of the formulation capable of preparing a clear AqED system was used to construct a ternary phase diagram using Origin software. Homogeneous and stable AqED systems were screened using the polymer dispersibility index (PDI) and centrifugal stability as indicators. Centrifugal stability test conditions: centrifugation at 8000 r / min for 10 min was performed, and the appearance and PDI before and after centrifugation were examined.

[0078] Preparation of EO-W: 10 μL of Platycladus orientalis leaf EO was added to an appropriate amount of water and vortexed to prepare a 1% (v / v) EO-W system.

[0079] The formulations that can form a clarified AqED system are shown in Table 2. The results were plotted using Origin software. Figure 6 The ternary phase diagram shown. Figure 6 The shaded areas represent the volume percentages of Platycladus orientalis leaf essential oil, eutectic solvent, and water, which, when mixed, are 0.5–30%, 29.5–70%, and 10–70%, respectively, forming a colorless, clear, and transparent AqED single-phase system that is non-greasy and easy to use. The unshaded areas represent the two-phase region, which appears cloudy. When the volume percentage of water is too small (less than 10%), the system is a viscous gel; when the water ratio is too high (greater than 70%), standing or centrifugation will cause emulsification or precipitation. Using PDI and centrifugal stability as indicators, the optimal formulation of homogeneous and stable AqED was screened (Table 3). The PDI of the 0.5% AqED, 1% AqED, and 2% AqED systems are all less than 0.3, indicating that the particle size in the AqED system is similar and the system distribution is relatively uniform. Figure 7 The centrifugal stability results of AqED are shown in Table 4 and... Figure 8 As shown, the EO-W system separates into layers after centrifugation because EO is oil-soluble but insoluble in water. In contrast, the 0.5% AqED, 1% AqED, and 2% AqED systems do not separate into layers after centrifugation, with a separation percentage (SP) value of 0 and a PDI value of less than 0.3 before and after centrifugation, indicating that the AqED system has good centrifugal stability.

[0080] Table 2: Clarification of the AqED System Prescription

[0081]

[0082] Table 3: Optimal formulations of DES, EO, and water for preparing AqED

[0083]

[0084] Table 4: Results of centrifugal stability study of AqED

[0085]

[0086] Example 4: Microscopic characterization of AqED

[0087] Raw materials were weighed according to the formula in Table 3, and 0.5% AqED, 1% AqED, and 2% AqED were prepared according to Example 3. Referring to the preparation method of AqED in Example 3, an appropriate amount of DES was taken, and an appropriate amount of water was added. The mixture was vortexed at room temperature and a rotation speed of 2800 r / min to prepare EO-free 0.5% AqED, 1% AqED, and 2% AqED. Specifically, in the EO-free 0.5% AqED, the volume percentages of DES and water were 31% and 69%, respectively; in the EO-free 1% AqED, the volume percentages of DES and water were 32% and 68%, respectively; and in the EO-free 2% AqED, the volume percentages of DES and water were 33% and 67%, respectively.

[0088] The hydrodynamic diameter of the AqED system was determined using the dynamic light scattering (DLS) method.

[0089] Take 10 μL of freshly prepared AqED, dilute it to 4 mL with deionized water, drop 10 μL onto a copper grid with a carbon support film, and after 20 min, use filter paper to absorb the excess solution from the edge of the copper grid. Negatively stain with 2% phosphotungstic acid solution, allow it to air dry at room temperature, and then observe the sample under a transmission electron microscope (TEM).

[0090] The DLS results are as follows: Figure 9 As shown, the AqED system without EO is a polydisperse system. The hydrodynamic diameters of the EO-free 0.5% AqED, 1% AqED, and 2% AqED systems are 11.20 nm, 8.55 nm, and 10.95 nm, respectively. After adding EO, the hydrodynamic diameters of the 0.5% AqED, 1% AqED, and 2% AqED systems increased to 301.18 nm, 507.85 nm, and 845.09 nm, respectively. DLS results indicate that the strong hydrophobic interactions and crystallization tendency in the AqED system cause the particles in the aqueous dispersion to aggregate and form enthalpy aggregates. TEM results are shown below. Figure 10 As shown, the particles in 0.5% and 1% AqED systems are irregularly shaped. In 2% AqED, in addition to the irregular particles, a distinct liquid crystal structure can also be observed. This indicates that when the EO content is low, the particle size in the AqED system is smaller, which is more conducive to transdermal delivery.

[0091] Example 5: Small-Angle X-ray Scattering Analysis by AqED

[0092] The internal microstructure of the AqED assembly was characterized using a small-angle X-ray scattering (SAXS) instrument.

[0093] Instrument conditions: copper target optical tube, tube voltage 40KV, tube current 40mA, wavelength 0.1542nm;

[0094] Detector: Imaging panel (0.07-28nm) -1 Decris Mythen2 1D (0.06-7nm) -1 );

[0095] Sample cell: Anton Paar's dedicated Paste Cell;

[0096] Testing Procedure: A small amount of DES prepared in Example 2, 1% AqED and 2% AqED prepared in Example 3 (formulas shown in Table 3) were taken and filled into Paste Cells respectively. After sealing, they were placed in the sample holder of the instrument and evacuated to 10°C. -5 After Pa, small-angle X-ray scattering was detected at 25℃ for 15-20 minutes. The detector recorded the scattering data, and finally a one-dimensional scattering curve was obtained.

[0097] The results are as follows Figure 11 As shown, a broad peak was detected in the low scattering vector q region of DES, indicating that DES does not possess a long-range ordered structure, but rather a nanoscale ordered structure. In the 1% AqED and 2% AqED systems, this peak was observed to shift towards the low q region, reaching a value close to 0.7 nm. -1 The appearance of a broad peak indicates that EO-DES undergoes self-aggregation in aqueous solution, forming a micelle phase. Example 6: Study on the formation mechanism of AqED

[0098] Through FTIR and 1 H-NMR method to investigate the binding mode of EO and DES.

[0099] Take appropriate amounts (5-10 mg) of samples of Platycladus orientalis leaf EO (prepared in Example 1), DES (prepared in Example 2), and EO-DES (prepared by vortexing 1 μL of Platycladus orientalis leaf EO and 32 μL of DES). Detect these samples using a TENSOR 27 FTIR spectrometer with a scanning range of 4000-450 cm⁻¹. -1 The scanning temperature was 25℃, the average number of scans was 20, the background was air, and the instrument resolution was 1.0 cm. -1 .

[0100] FTIR results as follows Figure 12 As shown in Figure A, compared with the infrared characteristic peaks of EO and DES, the infrared characteristic peaks of EO-DES showed different degrees of shift. The stretching vibration peak of the polymeric hydroxyl groups in EO-DES shifted from 3365.4 cm⁻¹. -1 The redshift reached 3361.3 cm. -1 The stretching vibration peak of the carbonyl C=O group is from 1692.4 cm⁻¹. -1 Redshifted to 1691.5cm -1 The changes in the infrared characteristic peaks indicate the presence of hydrogen bonds between EO and DES.

[0101] Take an appropriate amount of Platycladus orientalis leaf EO (prepared in Example 1), DES (prepared in Example 2), and EO-DES (prepared by vortexing 1 μL of Platycladus orientalis leaf EO and 32 μL of DES) samples (10-20 mg), dissolve them in 0.5 mL of DMSO-d6, and perform 1D characterization using an AVANCE AV-500 MHz spectrometer.

[0102] 1 H-NMR results are as follows Figure 12 As shown in Figure B, due to the presence of alcohols and phenols in EO, a significant phenolic hydroxyl peak is observed at δ 3.3 ppm. When EO and DES form EO-DES, the phenolic hydroxyl peak disappears, indicating that the binding of EO and DES differs from simple physical mixing. The hydrogen bond network between DES can adsorb complex components from Platycladus orientalis leaf EO, making the binding of EO and DES more stable.

[0103] The self-assembly mechanism of the AqED system was investigated using ultraviolet-visible absorption spectroscopy (UV-Vis). 10 μL of 1% AqED (prepared in Example 3, formulation shown in Table 3) was incubated for 30 min with 4 mL of water, 4 mL of DMSO, 4 mL of 0.2% (w / v) SDS solution, 4 mL of urea solution with gradient concentrations (0, 2, 4, 6 M), or 4 mL of NaCl solution with gradient concentrations (0, 0.25, 0.5, 1 M). The UV-Vis spectra of 1% AqED were recorded, with absorbance values ​​recorded in the range of λ200–800 nm.

[0104] UV-Vis results are as follows Figure 13 As shown in Figure A, Ch exhibits no absorption peak in the UV-Vis range, while the UV-Vis absorption spectra of Ge and DES show maximum absorbance at 228 nm. This can be attributed to the presence of α,β-unsaturated acids in the geranilic acid structure. Compared to the UV-Vis spectra of Ge and DES, the absorption peak of the 1% AqED system shows a blue shift. Figure 13 As shown in Figure B, the absorption peak of 1% AqED in DMSO changed significantly, with a red shift and decreased absorbance. This is because the main component of EO is a terpene compound, and the presence of double bonds in the structure allows EO to bind with DES through π-π stacking.

[0105] In addition, the UV-Vis spectra of 1% AqED treated with SDS, urea, or NaCl are as follows: Figure 13 C Figure 13 D、 Figure 13 As shown in Figure E, the absorption spectrum of 1% AqED red-shifted after the addition of SDS, indicating the presence of hydrophobic interactions between AqED molecules, possibly due to the involvement of SDS in the assembly of AqED. Urea's strong hydrophilicity gives it a certain dissociation ability, capable of disrupting intramolecular hydrogen bonds. NaCl can disrupt intermolecular electrostatic interactions. After the addition of urea or NaCl, the absorption spectrum of 1% AqED showed only a slight shift (…). Figure 13 D and Figure 13 E) indicates that the hydrogen bonding and electrostatic interactions between AqED molecules are not significant.

[0106] The above results indicate that the complex components in Platycladus orientalis leaf EO are adsorbed by the hydrogen bond network inside DES, and upon the addition of water, they self-assemble into an AqED system with a nanostructure. The self-assembly forces of AqED are mainly π-π stacking interactions and hydrophobic interactions. The hydrogen bond network in the AqED nanosystem can prevent the volatilization of EO and protect the components in EO from photothermal oxidation.

[0107] Example 7: In vitro skin penetration experiment of AqED

[0108] In vitro skin permeation experiments were conducted using the Franz diffusion cell. The lipid-soluble fluorescent probe fluorescein isothiocyanate (FITC) was used to simulate the in vitro skin permeation effect of the formulation AqED, with free FITC serving as a control. The percutaneous permeation of AqED was investigated. Twenty-four hours prior to the experiment, the fur on the backs of ICR mice was shaved. After euthanasia by dislocation, the skin on the backs of the mice was dissected, subcutaneous tissue was removed, and the skin was rinsed thoroughly with physiological saline, blotted dry with filter paper, and then placed between the receiving and supply cells of the diffusion cell, with the stratum corneum facing the supply cell.

[0109] Accurately weigh 1.5 mg of FITC, add 2.5 mL of PBS to dissolve, and prepare a FITC-labeled PBS solution (FITC concentration of 0.6 mg / mL).

[0110] Accurately weigh 1.5 mg of FITC and add 2.5 mL of 0.5% AqED, 1% AqED or 2% AqED (prepared in Example 3, the formulation is shown in Table 3) to dissolve it, so as to prepare a FITC-labeled 0.5%, 1% or 2% AqED solution (FITC concentration is 0.6 mg / mL).

[0111] PBS group: 500 μL of FITC-labeled PBS solution was evenly spread onto the skin epidermis. 0.5% AqED group, 1% AqED group, and 2% AqED group: FITC-labeled 0.5%, 1%, and 2% AqED solutions were evenly spread onto the skin epidermis, respectively. PBS solution containing 20% ​​ethanol was used as the receiving medium. At a temperature of 32 ± 0.5℃ and a stirring speed of 400 r / min, 1 mL of receiving medium was collected at 0.5, 1, 2, 4, 6, 8, 10, and 24 h, and an equal volume of receiving medium at the same temperature was added simultaneously. The fluorescence intensity of each sample was detected using a Cytation5 multimode microplate detection and cell imaging system. The cumulative permeation volume Q per unit area was calculated, and the kinetic curve of in vitro percutaneous permeation was plotted.

[0112]

[0113] Among them, Q(μg / cm 2 C represents the cumulative infiltration per unit area. n and C i Let V be the drug concentration in the receiving solution at the nth and ith sampling times, respectively; V be the volume of the diffusion cell (6.5 mL); Vi be the sampling volume at the i-th point (1 mL); and A be the effective permeation area of ​​the diffusion cell (2.2 cm²). 2 ).

[0114] Plotting the cumulative permeation rate Q against time t, the slope of the linear portion (between 0.5 and 12 hours) represents the steady-state transdermal rate J. ss (μg / cm 2 The point where the extended line of the out-of-phase graph intersects the horizontal axis is the residence time T. lag The permeability enhancement ratio ER is the J of each group. ss Compared with the control group J ss The ratio of .

[0115] like Figure 14 As shown in Figure A, the standard curve equation for the fluorescence intensity versus concentration of FITC is: F = 760.42C + 185.67, R 2=0.9999. Within the range of 0.1-200 ng / mL, the fluorescence intensity of FITC showed a linear correlation with concentration. The transdermal permeation curve and in vitro transdermal parameters over 24 hours are shown below. Figure 14 As shown in B and Table 5, the correlation coefficient r of the Qt linear regression equation within 0.5-12 hours of in vitro transdermal absorption... 2 Except for the 0.5% AqED group (0.9843), the values ​​for the other three groups were all greater than 0.99, indicating that the transdermal permeation behavior of each group basically conformed to the zero-order kinetic model and had a small dispersion range. Compared with the PBS group, the transdermal permeation capacity of AqED was significantly improved, and the transdermal absorption capacity was ranked as follows: 2% AqED > 1% AqED > 0.5% AqED.

[0116] Table 5: In vitro transdermal absorption parameters (n=3)

[0117]

[0118] Note: **P<0.01, ***P<0.0001 vs PBS group.

[0119] Example 8: In vivo skin penetration of AqED

[0120] Accurately weigh 0.3 mg of FITC, add 0.5 mL of PBS to dissolve, and prepare a FITC-labeled PBS solution (FITC concentration of 0.6 mg / mL).

[0121] Accurately weigh 0.3 mg FITC and add 0.5 mL of 1% AqED (prepared in Example 3, the formula is shown in Table 3) to dissolve it, thus preparing a FITC-labeled 1% AqED solution (FITC concentration is 0.6 mg / mL).

[0122] Six ICR mice were randomly divided into two groups according to body weight: a control group and a formulation group. Twenty-four hours before the experiment, a 2cm × 2cm smooth skin was shaved off the back of each mouse using electric clippers. The control group mice had 100μL of FITC-labeled PBS solution evenly applied to the shaved area, while the formulation group mice had 100μL of FITC-labeled 1% AqED solution evenly applied to the shaved area. After being kept in the dark for 2 hours, the mice were euthanized by cervical dislocation, and the skin of the shaved area was peeled off, removing the subcutaneous tissue. After rinsing with physiological saline, the stratum corneum (SC) side was placed upwards, and the samples were cut into strips approximately 1cm long and placed in a mold for cryo-embedding. The embedded samples were removed from the mold, cryosectioned, and sectioned, with a thickness controlled at approximately 10μm. The fluorescence transmittance in the tissue was observed using an upright fluorescence microscope, and the fluorescence intensity was semi-quantitatively analyzed using ImageJ software.

[0123] The results are as follows Figure 15As shown in Figure A, no significant fluorescence was observed in the dermis of the control group, with fluorescence accumulating in the SC. In contrast, significant fluorescence was observed in both the SC and dermis of the formulation group, indicating that 1% AqED can effectively penetrate the SC and enter the dermis. Semi-quantitative analysis of fluorescence intensity was performed using ImageJ software, and the results are as follows: Figure 15 As shown in Figure B, the fluorescence intensity of the formulation group was significantly higher than that of the control group. In vivo skin penetration studies demonstrated that AqED can effectively penetrate SC and deliver the drug to the dermis.

[0124] Example 9: Study on the transdermal mechanism of AqED

[0125] The transdermal mechanism of the AqED system was investigated using ATR FTIR.

[0126] Hair was shaved from the back of SD rats, and 1.5cm × 1.5cm pieces of skin were cut off. Subcutaneous tissue was removed, and the skin was rinsed with physiological saline and blotted dry with filter paper. The skin was then immersed in a 60°C water bath for 3 minutes to remove the surface layer, followed by overnight immersion in 0.25% (w / v) trypsin to separate the SC layer attached to the dermis. The SCs were thoroughly washed with distilled water and stored in a desiccator. Morphology of the SCs was observed using an upright fluorescence microscope. The SCs were washed in PBS and dried at room temperature for 72 hours. After ATR FTIR spectroscopy, the SCs were soaked in 500 μL of 1% or 2% AqED (prepared in Example 3, formulation shown in Table 3) for 24 hours, washed with PBS, dried at room temperature for 72 hours, and then subjected to a second ATR FTIR scan. The spectra before and after treatment were compared to evaluate the effect of AqED on the SCs.

[0127] The results are as follows Figure 16 As shown in Figure A, the separated SC sheet is intact, and the keratinocytes are arranged in a tightly packed, scale-like pattern. Figure 16 As shown in B, 2800-3000cm -1 The two characteristic peaks can be attributed to the absorbance of phospholipids and fatty acids. 2850 cm⁻¹ -1 and 2920cm -1 These are characteristic peaks generated by the symmetric and asymmetric vibrations of the -CH2 group of long-chain lipid hydrocarbons, respectively. Compared with SC slides treated with PBS, the peak at 2850 cm⁻¹ was significantly higher after treatment with the AqED system. -1 and 2920cm -1 The reduced peak area at the SC site leads to increased lipid mobility in the SC structure. AqED diffuses into the lipids, weakening lipid-lipid interactions and altering the lipid arrangement of the SC, thus enabling drug penetration into the skin. These results indicate that lipid extraction is a key mechanism for AqED transdermal delivery.

[0128] Example 10: Hair regrowth activity of AqED

[0129] Six ICR mice were randomly divided into two groups according to their body weight: a control group and a formulation group. Twenty-four hours before the experiment, smooth skin measuring 2cm × 2cm was shaved off the back of each mouse using electric clippers. From day 1 to day 4 after hair removal, the control group mice received 20μL of physiological saline evenly applied to the shaved area daily, while the formulation group mice received 20μL of 2% AqED (prepared in Example 3, formulation shown in Table 3) evenly applied to the shaved area daily. Subsequently, the dosage was adjusted to once every two days, with each dose remaining constant, until day 28.

[0130] Take photos daily to record the new hair growth in the treated area, and use ImageJ software to measure the coverage of new hair growth on day 14.

[0131]

[0132] The results are as follows Figure 17 and Figure 18 As shown, on day 14, the new hair coverage rate in the formulation group was 93.25±3.974%, significantly higher than that in the control group (0%); on day 16, the hair growth coverage rate in the formulation group reached 100%, while no hair growth occurred in the control group; on day 24, the new hair in the control group still did not completely cover the bald area; while after treatment with 2% AqED for 24 days, the hair grew to normal levels. The results indicate that AqED has significant hair regeneration promoting activity.

[0133] Example 11: Safety Evaluation of AqED

[0134] Twelve SD rats were randomly divided into four groups according to their body weight: saline group, EO group, DES group, and 1% AqED group, with three rats in each group.

[0135] Preparation of EO-jojoba oil: 5 μL of Platycladus orientalis leaf EO (prepared in Example 1) was dissolved in 495 μL of jojoba oil and vortexed to prepare EO-jojoba oil (EO volume percentage of 1%).

[0136] Preparation of DES-W: Dissolve 160 μL of DES (prepared in Example 2) in 340 μL of water, vortex mix well, and DES-W (32% by volume of DES) is prepared.

[0137] Twenty-four hours prior to the experiment, a 2cm × 2cm smooth skin strip was shaved off the back of each rat using electric clippers. Rats in the EO group received 150 μL of EO-jojoba oil evenly applied to the shaved area daily for 5 days; rats in the DES group received 150 μL of DES-W evenly applied to the shaved area daily for 5 days; rats in the 1% AqED group received 150 μL of 1% AqED (prepared in Example 3, formulation shown in Table 3) evenly applied to the shaved area daily for 5 days; and rats in the saline group received 150 μL of saline evenly applied to the shaved area daily for 5 days. Skin irritation reactions at the treatment sites were observed and recorded daily, and scores were assigned according to the scoring criteria tables (Tables 6 and 7) in the "Technical Guidelines for Research on Irritation, Allergy, and Hemolysis of Chemical Drugs" (No. 4). After administration, the skin tissue at the treatment sites was dissected, stained with H&E, and histologically observed to assess the skin irritation caused by the formulations.

[0138] Table 6: Skin Irritation Response Scoring Criteria

[0139]

[0140]

[0141] Table 7: Evaluation of Skin Irritation Intensity

[0142]

[0143] Table 8: Skin Irritation Reactions

[0144]

[0145] Table 9: Results of Skin Irritation Reactions

[0146]

[0147] The results are shown in Tables 8 and 9. The appearance of mild erythema in the DES group rats on day 4 indicates that DES has a mild irritant effect. No significant irritation was observed in the saline group, EO group, and 1% AqED group. H&E staining results are shown below. Figure 19 As shown, the DES group rats exhibited epidermal damage, including keratinocyte shedding (green arrows), significant keratinization (yellow arrows), and squamous epithelial hyperplasia (red arrows); dermal cell atrophy (green boxes) was observed in the dermis, accompanied by inflammatory cell infiltration (yellow boxes). Furthermore, the red boxes indicate that the cell morphology of the subcutaneous tissue in the DES group changed from spindle-shaped to ellipsoidal. These results suggest that DES has a mild irritant effect. Figure 19The black arrows indicate hair follicles in the skin tissue. Except for the DES group, where the hair follicles are still in the resting phase, the hair follicles in all other groups are in the growth phase. This indicates that both EO and AqED can accelerate the transition of hair follicles from the resting phase to the growth phase, promoting hair follicle cell proliferation. The AqED system can stably encapsulate EO, effectively penetrating the stratum corneum to enter the dermal hair follicle area, thereby exerting its hair regeneration-promoting activity.

Claims

1. A traditional Chinese medicine essential oil composition for treating hair loss, characterized in that: It includes a traditional Chinese medicine essential oil and a deep eutectic solvent, wherein the volume ratio of the traditional Chinese medicine essential oil to the deep eutectic solvent is 1:1 to 1:

80.

2. The herbal essential oil composition according to claim 1, characterized in that: The volume ratio of the herbal essential oil to the deep eutectic solvent is 1:1.6 to 1:62, preferably (0.5 to 2):(31 to 33), and more preferably (1 to 2):(32 to 33).

3. The herbal essential oil composition according to claim 1, characterized in that: The aforementioned Chinese herbal essential oil composition is obtained by adding Chinese herbal essential oil dropwise into a deep eutectic solvent and mixing them evenly.

4. A traditional Chinese medicine essential oil composition for treating hair loss, characterized in that: The aforementioned herbal essential oil composition is prepared by adding herbal essential oil droplets into a deep eutectic solvent, mixing them evenly, and then mixing them evenly with water to obtain an aqueous herbal essential oil transdermal delivery system.

5. The herbal essential oil composition according to claim 4, characterized in that: In the aforementioned transdermal delivery system for traditional Chinese medicine essential oils, the volume percentage of the traditional Chinese medicine essential oil is 0.5-30%, the volume percentage of the deep eutectic solvent is 29.5-70%, and the remainder is water; preferably, in the aforementioned transdermal delivery system for traditional Chinese medicine essential oils, the volume percentage of the traditional Chinese medicine essential oil is 0.5-2%, the volume percentage of the deep eutectic solvent is 31-33%, and the remainder is water; more preferably, in the aforementioned transdermal delivery system for traditional Chinese medicine essential oils, the volume percentage of the traditional Chinese medicine essential oil is 1-2%, the volume percentage of the deep eutectic solvent is 32-33%, and the remainder is water.

6. The traditional Chinese medicine essential oil composition according to claim 1 or 4, characterized in that: The herbal essential oil is selected from at least one of arborvitae leaf essential oil and rosemary essential oil, preferably arborvitae leaf essential oil.

7. The traditional Chinese medicine essential oil composition according to claim 1 or 4, characterized in that: The deep eutectic solvent is a low eutectic mixture formed by hydrogen bond donors and hydrogen bond acceptors, wherein the molar ratio of hydrogen bond acceptors to hydrogen bond donors is 1:2; the hydrogen bond donors are selected from at least one of organic acids, polyols and sugars; and the hydrogen bond acceptors are selected from at least one of quaternary ammonium salts and zwitterionic surfactants.

8. The herbal essential oil composition according to claim 7, characterized in that: The hydrogen bond donor is geraniol, and the hydrogen bond acceptor is choline or its bicarbonate.

9. A method for preparing the traditional Chinese medicine essential oil composition according to claim 4, comprising: The essential oil of traditional Chinese medicine was added dropwise to a deep eutectic solvent, vortexed and mixed evenly, and then mixed evenly with water to prepare an aqueous transdermal delivery system for the essential oil of traditional Chinese medicine.

10. The use of the herbal essential oil composition according to claim 1 or 4 in the preparation of a medicament with hair regeneration-promoting activity, preferably in the preparation of a medicament for treating hair loss, and more preferably in the preparation of a medicament for treating androgenetic alopecia.

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