Baicalein liquid preparation, preparation method and application thereof

Abstract

The application discloses a baicalein liquid preparation and a preparation method and application thereof, and belongs to the technical field of cosmetic and daily chemical active material preparation. In view of the problems of poor water solubility and stability of high-purity baicalein and high cost and unsuitability of existing solubilization technology for the cosmetic field, the baicalein is first extracted from the root of Scutellaria baicalensis Georgi, the baicalin is subjected to acid hydrolysis to obtain a baicalein crude product, then the baicalein crude product is subjected to silica gel chromatography purification, fractions with a baicalein content of 40-60% are collected, finally, the obtained baicalein is mixed with polyols and a stabilizer to prepare a clear and transparent water-soluble solution. The defects of poor water solubility, easy precipitation of high-purity baicalein, low effective component content of a crude extract and deep color are overcome, the obtained water-soluble baicalein solution has good stability, no precipitation occurs after 12 months of storage at room temperature, the solution remains clear after being diluted by 50 times of water, and the water-soluble baicalein solution has high effective component content and light color, and is especially suitable for the cosmetic and daily chemical fields.

Description

Technical Field

[0001] This invention belongs to the field of preparation technology of active raw materials for cosmetics and daily chemical products, and specifically relates to the preparation and application of a water-soluble baicalein solution. Background Technology

[0002] Baicalein is one of the main flavonoid active components in the root of Scutellaria baicalensis Georgi. It possesses various biological activities, including antioxidant, anti-inflammatory, antibacterial, and anti-allergic effects, and has broad application prospects in the fields of cosmetics and daily chemical products. However, baicalein itself is a non-water-soluble component with extremely low solubility in water (micrograms / ml), making it difficult to formulate stable water-based cosmetic formulations directly.

[0003] In existing technologies, methods to improve the water solubility of baicalin mainly include cyclodextrin inclusion, preparation of nanolipid carriers, and chemical modification. For example, the invention patent with publication number CN115557924A uses cyclodextrin to form an inclusion complex eutectic with baicalin, achieving better water solubility and thermal stability. However, cyclodextrin inclusion technology has problems such as limited inclusion efficiency, low drug loading, and high cost, making it difficult to meet the dual requirements of the cosmetic industry for active ingredient content and cost control. Another example is the invention patent with publication number CN110051654A, which uses N-trimethyl chitosan-modified lipid nanoparticles to encapsulate baicalin for ophthalmic formulations. This formulation has strong drug loading capacity and good biocompatibility, but its preparation process is complex, involves organic solvents, and is costly. Furthermore, the stability of the nanosystem under long-term storage and dilution conditions remains a challenge, making it unsuitable for large-scale cosmetic production.

[0004] On the other hand, in the field of baicalin raw material production, existing patents mostly focus on the industrial preparation of high-purity baicalin monomer powder. For example, the invention patent with publication number CN103160549A uses endogenous enzyme hydrolysis, negative pressure cavitation extraction, normal-phase silica gel low-pressure preparative liquid chromatography, low-temperature crystallization and recrystallization to obtain high-purity baicalin with a purity of over 98%. Similarly, traditional baicalin extraction processes generally employ unit operations such as water / alcohol extraction, acid precipitation, macroporous adsorption resin chromatography, high-concentration strong acid high-temperature catalytic hydrolysis, low-temperature cold crystallization, and activated carbon / clay decolorization and purification. The target product is high-purity (>95%) crystalline powder, mainly targeting the pharmaceutical raw material field. This type of high-purity baicalin has a regular crystal structure and high lattice energy. Even with temporary dissolution with the help of additives, it is very easy to recrystallize after refrigeration and dilution with water, making it completely unsuitable for long-term stable storage and formulation of water-based cosmetics. Meanwhile, crude baicalein extracts that have not undergone fine purification in the industry, although they do not have obvious crystallization problems, have low content of active ingredients, numerous impurities, and a dark brown color, which cannot meet the requirements of cosmetics for active ingredient content, appearance color, and compatibility stability.

[0005] In summary, existing technologies face a significant dilemma: high-purity baicalin meets the required content but exhibits extremely poor water solubility and is prone to crystallization; while crude extracts show anti-crystallization effects, their content is low and their color is poor. Existing solubilization technologies (cyclodextrin inclusion complexation, nanocarriers, and chemical modification) can improve water solubility to some extent, but they suffer from high costs, complex processes, low drug loading, and insufficient stability. Furthermore, they are primarily designed for pharmaceutical injection or oral administration routes and are not suitable for the large-scale production needs of water-based cosmetic formulations.

[0006] Therefore, developing a preparation process for a baicalein-specific daily chemical raw material that can maintain the natural activity of baicalein, have good water solubility and long-term storage stability, and simultaneously take into account the appropriate content of effective ingredients and light color, has important practical application value. Summary of the Invention

[0007] This invention addresses the technical problems of poor water solubility and stability of high-purity baicalein, as well as the high cost and unsuitability of existing solubilization technologies for cosmetic processes. It proposes a liquid baicalein preparation, its preparation method, and its application.

[0008] To achieve the above objectives, the technical solution of the present invention is implemented as follows:

[0009] This invention provides a method for preparing a liquid baicalin formulation, comprising the following steps:

[0010] (1) Add water to the crude powder of Scutellaria baicalensis root and decoct twice. Combine the two filtrates and add hydrochloric acid to adjust the pH. After standing, centrifuge. Add water to the precipitate, then add sodium hydroxide solution to adjust the pH and add ethanol solution for filtration. Adjust the pH of the filtrate again and let it stand. Recrystallize the precipitate obtained by filtration to obtain baicalin.

[0011] (2) Dissolve the baicalin obtained in step (1) in hydrochloric acid solution and perform water bath reflux hydrolysis. Monitor the reaction endpoint with TLC. After the reaction is completed, adjust the pH of the solution. After extraction and concentration, crude baicalin is obtained. After silica gel chromatography purification, the desired baicalin concentrate is obtained.

[0012] (3) Add the baicalin enrichment obtained in step (2) to a polyol and stir to disperse it. Then add a stabilizer, continue stirring to homogenize, and then add another polyol. After heating and stirring to age, the baicalin liquid preparation is obtained.

[0013] In step (1), the mass ratio of water to coarse Scutellaria baicalensis root powder is 8-12:1, preferably 10:1, and the decoction time is 0.5-2h, preferably 1h.

[0014] The first pH adjustment is 1-2; the second pH adjustment uses a sodium hydroxide solution with a mass percentage of 40% and adjusts the pH to 6.5-7.5, preferably 7; the third pH adjustment is 1-2; the ethanol solution has a mass percentage of 40-60%; and the recrystallization is performed using 40%-60% ethanol, preferably 50% ethanol.

[0015] In step (2), the hydrochloric acid solution has a mass percentage of 2-4%. Based on 1g of baicalin, 10-20mL of hydrochloric acid solution is added to each gram of baicalin.

[0016] The water bath reflux temperature is 90-100℃, preferably 100℃, and the time is 0.5-2 seconds. The developing solvent for the TLC monitoring reaction is petroleum ether:ethyl acetate = 1:2, and the endpoint is the complete disappearance of the baicalin spots. The pH of the adjusted solution is 6-7.

[0017] The conditions for silica gel chromatography are as follows: silica gel 300-400 mesh, silica gel amount (mass) is 8-15 times the sample amount (mass), preferably 10 times, eluent is petroleum ether: ethyl acetate = 4:1-1:1 gradient elution, and fractions with Rf value of 0.1-0.2 are collected by TLC monitoring.

[0018] In step (3), the polyol is selected from at least one of 1,3-propanediol, butanediol, methylpropanediol, glycerol and dipropylene glycol. The high proportion of polyol in the system can form intermolecular hydrogen bonds with the phenolic hydroxyl groups of baicalein, while regulating the polarity of the mixed solvent. Based on the principle of like dissolves like, the molecular-level solubility of baicalein is improved, the tendency of baicalein molecules to aggregate is weakened, and the driving force for crystallization is reduced from a thermodynamic perspective. The stabilizer is selected from at least one of Tween 20, Tween-80, lecithin, polyethylene glycol, AEO-3, AEO-7, AEO-9, poloxamer, and ppg-5 cetyl ether-20. The total added mass of the polyol and stabilizer is ≥ 5% of the total mass of the system, and the mass ratio of the polyol to the stabilizer is 1:4-3:2. After the compounded nonionic stabilizer reaches the critical micelle concentration, it forms a micelle structure with hydrophilic exterior and lipophilic interior, which encapsulates the lipophilic baicalein in the micelle core, thereby achieving micelle solubilization. At the same time, the stabilizer molecules are adsorbed on the surface of microcrystals, forming steric hindrance, which hinders particle collision and aggregation and crystal growth, and is resistant to temperature changes and high dilution without breaking stability.

[0019] The polyol is added as follows: 40% is added first to disperse baicalein, and then the remaining 60% is added; the homogenization time is 2-3 hours, and the temperature is 25-80℃; the aging time is ≥8 hours, and the temperature is 20-80℃. The segmented polyol feeding method achieves thorough wetting and deagglomeration of the powder, and long-term homogenization breaks down tiny particles; the long-term heating and aging process causes unstable microcrystals in the system to redissolve, the molecular distribution to become more uniform, and potential crystal nuclei to be eliminated in advance, enabling the system to reach a state of thermodynamic and kinetic stability. This ensures that no precipitation occurs during long-term storage at room temperature, refrigeration, and even after 50-fold dilution.

[0020] This invention provides a liquid baicalein preparation obtained using the aforementioned preparation method. This invention limits the purity of baicalein to 40%-60%, retaining appropriate amounts of naturally occurring associated flavonoids and phenolic derivatives in the composition. These substances can embed into the baicalein molecular lattice, disrupting the regular arrangement and directional stacking of baicalein molecules, inhibiting crystal nucleus formation and crystal growth. This avoids the defects of high-purity baicalein (above 95%) having a regular lattice and being extremely prone to crystallization, while also avoiding the problems of excessive impurities and excessively dark color in low-purity crude extracts, forming a unique crystal-inhibiting balance system.

[0021] The present invention also provides the application of the baicalin liquid formulation in cosmetics or daily chemical products.

[0022] The beneficial effects of this invention are:

[0023] 1. This invention employs silica gel chromatography to directionally enrich 40%-60% of the intermediate purity component. The obtained baicalin is then mixed with polyols and stabilizers to prepare a clear and transparent water-soluble solution. This overcomes the technical challenges of high-purity baicalin's tendency to crystallize and the low content and poor color of crude extracts. Through a multi-pronged approach of specific purity crystal inhibition, polyol solubilization, micellar solubilization, and process aging for crystal stabilization, unexpected stabilization effects are achieved. The process utilizes conventional unit operations such as water decoction, acid precipitation, alcohol precipitation, silica gel chromatography, and compound homogenization. The raw materials are readily available, equipment requirements are low, and the process is environmentally friendly, making it suitable for large-scale industrial production.

[0024] 2. The formulation obtained by this invention is a clear and transparent liquid. It shows no precipitation, no sedimentation, and minimal color change after 12 months of storage at room temperature away from light, and retains a high baicalin content. Even after 50-fold dilution with water, it maintains a homogeneous and clear state, making it suitable for various water-based cosmetic formulations. It is convenient to add and use, imparting antioxidant and anti-inflammatory effects to products without affecting the clarity or storage stability of the cosmetic system. Attached Figure Description

[0025] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0026] Figure 1 The simulation equation is for the baicalin liquid formulation sample prepared in Example 1.

[0027] Figure 2 The effect of the sample on the viability of HaCaT cells.

[0028] Figure 3 The effect of the sample on IL-1α induced by TNF-α and IFN-γ in HaCaT cells.

[0029] Figure 4 The effect of the sample on IL-6 in TNF-α and IFN-γ induced HaCaT cells.

[0030] Figure 5 The effect of the sample on CCL17 in HaCaT cells induced by TNF-α and IFN-γ.

[0031] Figure 6 The effect of the sample on FLG in HaCaT cells induced by TNF-α and IFN-γ. Detailed Implementation

[0032] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0033] Example 1

[0034] A method for preparing a liquid baicalein formulation, the specific steps of which are as follows:

[0035] (1) Preparation of baicalin: Take 1 kg of crude powder of Scutellaria baicalensis root, add 10 L of water and decoct for 1 hour, then filter; add 10 L of water to the residue and decoct for 1 hour, then filter, and combine the two filtrates; add hydrochloric acid to adjust the pH of the filtrate to 1.5, let stand overnight, centrifuge and collect the precipitate; add an appropriate amount of water to the precipitate and stir well, adjust the pH to 7 with 40% NaOH, add an equal volume of ethanol, and filter; add hydrochloric acid to adjust the pH of the filtrate to 1.5, stir thoroughly, let stand overnight, filter and collect the precipitate; recrystallize the precipitate with 50% ethanol to obtain about 90 g of baicalin with an HPLC purity of about 85.6%.

[0036] (2) Preparation of crude baicalin: Take 50g of baicalin obtained in step (1), add 750mL of 2.5% hydrochloric acid solution (solid-to-liquid ratio 1:15), reflux hydrolyze, monitor by TLC (developing solvent petroleum ether: ethyl acetate = 1:2) until the baicalin spots completely disappear (about 1.5 hours); cool to room temperature, neutralize to pH 6.5 with 10% NaOH; extract with ethyl acetate 3 times, 300mL each time, combine the organic phases, concentrate under reduced pressure to dryness, and obtain about 78g of crude baicalin.

[0037] (3) Silica gel chromatography purification: Take 30g of crude baicalein obtained in step (2), mix it with 300g of silica gel (300 mesh), and dry sample loading; elute with petroleum ether: ethyl acetate = 4:1 gradient to 1:1, monitor by TLC (developing solvent petroleum ether: ethyl acetate = 1:2), collect fractions with Rf values ​​of about 0.1-0.2; combine the collected liquids, concentrate to dryness under reduced pressure, and obtain about 60g of baicalein enrichment. The baicalein content was determined to be 56.9% by HPLC external standard method.

[0038] (4) Preparation of baicalein liquid preparation: Take 2.0g of the baicalein enrichment (baicalein content 56.9%) obtained in step (3), add 40g of 1,3-propanediol, stir and disperse, add 5g of stabilizer ppg-5 cetyl ether-20, stir and homogenize at room temperature for 2.5 hours; add the remaining 60g of 1,3-propanediol, heat and stir to age for 8 hours, and obtain a transparent and clear yellowish-brown liquid, which is the baicalein liquid preparation (number 201); the total system mass is about 107g, of which the total mass of polyol is 100g (accounting for 93.5%), and the stabilizer accounts for 4.7%; by HPLC determination, the actual content of baicalein in the solution is 1.17%.

[0039] Example 2

[0040] A method for preparing a liquid baicalein formulation, the specific steps of which are as follows:

[0041] (1) Preparation of baicalin: Take 1 kg of crude powder of Scutellaria baicalensis root, add 10 L of water and decoct for 1 hour, then filter; add 10 L of water to the residue and decoct for 1 hour, then filter, and combine the two filtrates; add hydrochloric acid to adjust the pH of the filtrate to 1.5, let stand overnight, centrifuge to collect the precipitate; add an appropriate amount of water to the precipitate and stir well, adjust the pH to 7 with 40% NaOH, add an equal volume of ethanol, and filter; add hydrochloric acid to adjust the pH of the filtrate to 1.5, stir thoroughly, let stand overnight, filter to collect the precipitate; recrystallize the precipitate with 50% ethanol to obtain about 82 g of baicalin with an HPLC purity of 84%.

[0042] (2) Preparation of crude baicalin: Take 50g of baicalin obtained in step (1), add 750mL of 2.5% hydrochloric acid solution (material-liquid ratio 1:15), reflux hydrolyze, monitor by TLC (developing solvent petroleum ether: ethyl acetate = 1:2) until the baicalin spots completely disappear (about 1.5 hours); cool to room temperature, neutralize to pH 6.5 with 10% NaOH; extract with ethyl acetate 3 times, 300mL each time, combine the organic phases, concentrate under reduced pressure to dryness, and obtain about 77g of crude baicalin.

[0043] (3) Silica gel chromatography purification: Take 30g of crude baicalein obtained in step (2), mix it with 300g of silica gel (300 mesh), and dry sample loading; elute with petroleum ether: ethyl acetate = 4:1 gradient to 1:1, monitor by TLC (developing solvent petroleum ether: ethyl acetate = 1:2), collect fractions with Rf values ​​of about 0.1-0.2; combine the collected liquids, concentrate to dryness under reduced pressure, and obtain about 52g of baicalein concentrate. The baicalein content was determined to be 50.1% by HPLC external standard method.

[0044] (4) Preparation of baicalin liquid preparation: Take 2.0g of the baicalin enrichment (baicalin content 50.1%) obtained in step (3), add 40g of 1,3-propanediol, stir and disperse, add 5g of stabilizer Tween-80, stir and homogenize at room temperature for 2.5 hours; add the remaining 60g of 1,3-propanediol, heat and stir to age for 8 hours, and obtain a transparent and clear yellowish-brown liquid; the total mass of the system is about 107g, of which the total mass of polyol is 100g (accounting for 93.5%) and the stabilizer accounts for 4.7%; according to HPLC, the actual content of baicalin in the solution is 0.9%.

[0045] Example 3

[0046] A method for preparing a liquid baicalein formulation, the specific steps of which are as follows:

[0047] (1) Preparation of baicalin: Take 1 kg of crude powder of Scutellaria baicalensis root, add 8 L of water and decoct for 2 hours, then filter; add 8 L of water to the residue and decoct for 1 hour, then filter, and combine the two filtrates; add hydrochloric acid to adjust the pH of the filtrate to 1, let stand overnight, centrifuge and collect the precipitate; add an appropriate amount of water to the precipitate and stir well, adjust the pH to 6.5 with 40% NaOH, add an equal volume of ethanol, and filter; add hydrochloric acid to adjust the pH of the filtrate to 1, stir thoroughly, let stand overnight, filter and collect the precipitate; recrystallize the precipitate with 40% ethanol to obtain about 72 g of baicalin with an HPLC purity of 83%.

[0048] (2) Preparation of crude baicalin: Take 50g of baicalin obtained in step (1), add 1000mL of 2% hydrochloric acid solution (material-liquid ratio 1:20), reflux hydrolyze, monitor by TLC (developing solvent petroleum ether: ethyl acetate = 1:2) until the baicalin spots completely disappear (about 1.5 hours); cool to room temperature, neutralize to pH 6 with 10% NaOH; extract with ethyl acetate 3 times, 300mL each time, combine the organic phases, concentrate under reduced pressure to dryness, and obtain about 61g of crude baicalin.

[0049] (3) Silica gel chromatography purification: Take 30g of crude baicalein obtained in step (2), mix it with 450g of silica gel (400 mesh), and dry sample loading; elute with petroleum ether: ethyl acetate = 4:1 gradient to 1:1, monitor by TLC (developing solvent petroleum ether: ethyl acetate = 1:2), collect fractions with Rf values ​​of about 0.1-0.2; combine the collected liquids, concentrate to dryness under reduced pressure, and obtain about 48g of baicalein concentrate. The baicalein content was determined to be 47.1% by HPLC external standard method.

[0050] (4) Preparation of baicalin liquid preparation: Take 2.0g of the baicalin concentrate obtained in step (3), add 40g of methyl propylene glycol, stir and disperse, add 5g of stabilizer polyethylene glycol, stir and homogenize at room temperature for 2 hours; add the remaining 60g of methyl propylene glycol, heat and stir to age for 10 hours, and obtain a transparent and clear yellowish-brown liquid, which is the baicalin liquid preparation; the total system mass is about 107g, of which the total mass of polyol is 100g (accounting for 93.5%), and the stabilizer accounts for 4.7%; according to HPLC, the actual content of baicalin in the solution is 1.12%.

[0051] Example 4

[0052] A method for preparing a liquid baicalein formulation, the specific steps of which are as follows:

[0053] (1) Preparation of baicalin: Take 1 kg of crude powder of Scutellaria baicalensis root, add 12 L of water and decoct for 0.5 hours, then filter; add 12 L of water to the residue and decoct for 0.5 hours, then filter, and combine the two filtrates; add hydrochloric acid to adjust the pH of the filtrate to 2, let stand overnight, centrifuge and collect the precipitate; add an appropriate amount of water to the precipitate and stir well, adjust the pH to 7.5 with 40% NaOH, add an equal volume of ethanol, and filter; add hydrochloric acid to adjust the pH of the filtrate to 2, stir thoroughly, let stand overnight, filter and collect the precipitate; recrystallize the precipitate with 60% ethanol to obtain about 68 g of baicalin with an HPLC purity of 81%.

[0054] (2) Preparation of crude baicalin: Take 50g of baicalin obtained in step (1), add 500mL of 4% hydrochloric acid solution (material-liquid ratio 1:10), reflux hydrolyze, monitor by TLC (developing solvent petroleum ether: ethyl acetate = 1:2) until the baicalin spots completely disappear (about 1.5 hours); cool to room temperature, neutralize to pH 7 with 10% NaOH; extract with ethyl acetate 3 times, 300mL each time, combine the organic phases, concentrate under reduced pressure to dryness, and obtain about 55g of crude baicalin.

[0055] (3) Silica gel chromatography purification: Take 30g of crude baicalein obtained in step (2), mix it with 240g of silica gel (400 mesh), and dry sample loading; elute with petroleum ether: ethyl acetate = 4:1 gradient to 1:1, monitor by TLC (developing solvent petroleum ether: ethyl acetate = 1:2), collect fractions with Rf values ​​of about 0.1-0.2; combine the collected liquids, concentrate to dryness under reduced pressure, and obtain about 37g of baicalein enrichment. The baicalein content was determined to be 42% by HPLC external standard method.

[0056] (4) Preparation of baicalein liquid preparation: Take 2.0g of baicalein concentrate obtained in step (3), add 40g of dipropylene glycol, stir and disperse, add 5g of stabilizer AEO-7, stir and homogenize at 80℃ for 3 hours; add the remaining 60g of dipropylene glycol, heat and stir to age for 12 hours, and obtain a transparent and clear yellowish-brown liquid, which is the baicalein liquid preparation; the total system mass is about 107g, of which the total mass of polyol is 100g (accounting for 93.5%), and the stabilizer accounts for 4.7%; by HPLC determination, the actual content of baicalein in the solution is 1.07%.

[0057] Comparative Example 1

[0058] A method for preparing a high-purity baicalein preparation, the specific steps of which are as follows:

[0059] Take 2.0g of commercially available high-purity baicalein (HPLC baicalein content 95%), add 40g of 1,3-propanediol, stir and disperse, add 5g of stabilizer ppg-5 cetyl ether-20, stir and homogenize at room temperature for 2.5 hours; add the remaining 60g of 1,3-propanediol, heat and stir for aging for 8 hours. Results: It can be dissolved by heating to obtain a clear solution, but after being refrigerated at 4℃ for 24 hours, a large number of crystals precipitate; the solution dissolved by heating is diluted with water at a ratio of 1:50, and a white solid precipitates immediately.

[0060] Comparative Example 2

[0061] A method for preparing a crude baicalein preparation, the specific steps of which are as follows:

[0062] (1) Preparation of baicalin: Take 1 kg of crude powder of Scutellaria baicalensis root, add 10 L of water and decoct for 1 hour, then filter; add 10 L of water to the residue and decoct for 1 hour, then filter, and combine the two filtrates; add hydrochloric acid to adjust the pH of the filtrate to 1.5, let stand overnight, centrifuge and collect the precipitate; add an appropriate amount of water to the precipitate and stir well, adjust the pH to 7 with 40% NaOH, add an equal volume of ethanol, and filter; add hydrochloric acid to adjust the pH of the filtrate to 1.5, stir thoroughly, let stand overnight, filter and collect the precipitate; recrystallize the precipitate with 50% ethanol to obtain about 80 g of baicalin with an HPLC purity of about 95%.

[0063] (2) Preparation of crude baicalin: Take 50g of baicalin obtained in step (1), add 750mL of 2.5% hydrochloric acid solution (solid-to-liquid ratio 1:15), reflux hydrolyze, monitor by TLC (developing solvent petroleum ether: ethyl acetate = 1:2) until the baicalin spots completely disappear (about 1.5 hours); cool to room temperature, neutralize to pH 6.5 with 10% NaOH; extract with ethyl acetate 3 times, 300mL each time, combine the organic phases, concentrate under reduced pressure to dryness, and obtain about 32g of crude baicalin.

[0064] (3) Preparation of baicalin liquid preparation: Take 2.0g of crude baicalin obtained in step (2), add 40g of 1,3-propanediol, stir and disperse, add 5g of stabilizer ppg-5 cetyl ether-20, stir and homogenize at room temperature for 2.5 hours; add the remaining 60g of 1,3-propanediol, heat and stir to age for 8 hours, and obtain a clear liquid with a total solids concentration of 2%; however, HPLC determination shows that the actual content of baicalin in the liquid is <0.5% (calculated based on the content of baicalin in the crude product being about 25%), and the liquid color is very dark (dark brown), which is not suitable for cosmetic application.

[0065] Example of implementation effect 1

[0066] The baicalin content of the baicalin enrichment prepared in Example 1 was determined, and the specific steps are as follows:

[0067] 1. Experimental Instruments: Agilent ZORBAX SB-C18 (4.6 mm × 250 mm, 5 μm) chromatographic column

[0068] 2. Experimental reagents: Acetonitrile, HPLC grade

[0069] 3. Test conditions:

[0070] Mobile phase:

[0071] Phase A: Acetonitrile

[0072] Phase B: 0.1% phosphoric acid aqueous solution

[0073] Elution gradient: (0-50 min, 35%A → 55%A; 55-60 min, 55%A → 95%A; 60-70 min, 95%A; 70-73 min, 35%A; 73-80 min, 35%A)

[0074] Detection wavelength: 278nm

[0075] Flow rate: 1.0 mL / min

[0076] Column temperature: 25℃

[0077] Injection volume: 10 μL

[0078] 4. Experimental Procedure:

[0079] Plotting the standard curve:

[0080] Accurately weigh 0.01 g (accurate to 0.0001 g) of baicalin standard sample into a 50 mL beaker, dissolve in an appropriate amount of methanol, and dilute to a 50 mL volumetric flask to obtain a 0.2 mg / mL baicalin standard solution. Transfer 2.5 mL and 5.0 mL of this standard solution sequentially into 10 mL volumetric flasks, and dilute to a 10 mL volumetric flask to obtain standard solutions with concentrations of 0.05 and 0.10 mg / mL. Filter the 0.05, 0.10, and 0.2 mg / mL standard solutions through a 0.45 μm filter membrane, and reserve the filtrate.

[0081] Accurately pipette 10 μL of standard solution (concentrations of 0.05, 0.10, and 0.2 mg / mL) into HPLC to obtain the absorption peak area. Plot a standard curve based on the concentration and absorption peak area, and simulate the standard linear equation.

[0082] Sample content testing:

[0083] Accurately weigh 0.01 g (accurate to 0.0001 g) of baicalin sample into a 50 mL beaker, dissolve it in an appropriate amount of methanol, and dilute to a 50 mL volumetric flask to obtain a 0.2 mg / mL baicalin sample solution. Filter through a 0.45 μm filter membrane; the filtrate is the test solution.

[0084] A precise 10 μL sample was injected for testing, and the absorption peak area was obtained. The test was repeated twice, and the average value was taken. The corresponding test concentration was obtained according to the simulation equation. The test concentration divided by the concentration of the baicalin test sample (approximately 0.2 mg / mL) yielded the percentage content of baicalin. The simulation equation for the sample in Example 1 is as follows: Figure 1As shown, the concentration of the test sample was 0.218 mg / mL, and the average integral area of ​​the test sample was 6825. Substituting these values ​​into the equation, the concentration of the test sample was 0.1241 mg / mL, and the corresponding percentage concentration was (0.1241 / 0.218) × 100% = 56.9%.

[0085] The test results of Examples 1-4 are shown in Table 1:

[0086] Table 1. Results of baicalin determination in the baicalin enrichments prepared in Examples 1-4

[0087]

[0088] Example of implementation effect 2

[0089] The stability of the baicalin liquid formulation prepared in Example 1 was tested, as follows:

[0090] The baicalin liquid preparation (batch number 201) prepared in Example 1 was stored at room temperature (25±2℃) in the dark for 12 months, and its appearance was observed and the baicalin content was determined periodically. The results are as follows:

[0091] Appearance: It remains transparent and clear throughout, without any sediment or precipitation, and the color does not change significantly.

[0092] Content: Initially 1.17%, after 12 months 1.15%, content retention rate >98%.

[0093] Dilution stability: The solution was diluted with water at a ratio of 1:50 (v / v) to obtain a clear, pale yellow, transparent solution without any precipitation.

[0094] Example of implementation effect 3

[0095] The effects of the baicalin liquid preparation prepared in Example 1 on keratinocytes were analyzed, as follows:

[0096] (1) Sample preparation

[0097] The samples (oat alkaloids, purslane extract, baicalin liquid preparation, and centella asiatica extract) were diluted 100, 200, or 500 times with culture medium for experiments, and were divided into high, medium, and low dose groups (oat alkaloids: DMS-H, DMS-M, DMS-L); high, medium, and low dose groups of purslane extract (MCX-H, MCX-M, MCX-L); high, medium, and low dose groups of baicalin liquid preparation (HQ-H, HQ-M, HQ-L); and high, medium, and low dose groups of centella asiatica extract (JXC-H, JXC-M, JXC-L).

[0098] (2) Cytotoxicity test

[0099] Cell viability was assessed using the 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfonic acid benzene)-2H-tetrazole monosodium salt cell proliferation and toxicity assay kit (CCK-8). HaCaT cells were seeded in 96-well plates, and different concentrations of samples were added and incubated for 24 h. Subsequently, 10 μL of CCK-8 was added to each well and the plates were incubated at 37°C and 5% CO2 for 3 h. The absorbance was measured at 450 nm using a microplate reader.

[0100] The cell survival rate (%) is calculated as follows: Cell survival rate (%) = (experimental wells - blank wells) / (control wells - blank wells) × 100%.

[0101] The results are as follows Figure 2 As shown, oat alkaloids (DMS) at medium and high concentrations, with an action time of 24 hours, significantly inhibited the viability of HaCaT cells in a concentration-dependent manner, while the other three samples had no effect on HaCaT cell viability at any concentration. Therefore, subsequent experiments mainly focused on three samples: baicalein liquid preparation, purslane extract, and centella asiatica extract.

[0102] (3) qPCR detection

[0103] HaCaT cells were treated with different concentrations of samples for 24 hours, followed by treatment with 10 ng / mL TNF-α and IFN-γ for 8 hours. The cells were then frozen using Trizol, or total RNA was directly extracted and reverse transcribed into cDNA. The cDNA was then detected using Novizan's SYBR qPCR Master Mix. The relative gene expression was calculated using the 2-ΔΔCT method.

[0104] The results are as follows Figure 3 and Figure 4 As shown, Scutellaria baicalensis root, Portulaca oleracea and Centella asiatica can all significantly inhibit the mRNA expression of IL-1α and IL-6 in HaCaT cells induced by TNF-α and IFN-γ. Among them, the liquid preparation of baicalein has the most significant effect and is concentration-dependent.

[0105] Figure 5 The results of the samples on the effects of HaCaT cell chemokines show that only the baicalin liquid preparation can significantly inhibit the expression of CCL17 mRNA in HaCaT cells induced by TNF-α and IFN-γ in a concentration-dependent manner.

[0106] Figure 6The study investigated the effects of the samples on filaggrin in HaCaT cells. It was found that only the baicalin liquid preparation could inhibit the mRNA expression of filaggrin (FLG) in HaCaT cells induced by TNF-α and IFN-γ in a concentration-dependent manner.

[0107] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for preparing a liquid baicalin formulation, characterized in that, Includes the following steps: (1) Add water to the crude powder of Scutellaria baicalensis root and decoct twice. Combine the two filtrates and adjust the pH. After standing, centrifuge. Add water to the precipitate, adjust the pH and add ethanol solution for filtration. Adjust the pH of the filtrate again and let it stand. Recrystallize the precipitate obtained by filtration to obtain baicalin. (2) Dissolve the baicalin obtained in step (1) in hydrochloric acid solution and perform water bath reflux hydrolysis. After the hydrolysis is completed, adjust the pH of the solution. After extraction and concentration, crude baicalin is obtained. It is then purified by silica gel chromatography. The silica gel chromatography conditions are: silica gel 300-400 mesh, silica gel amount is 8-15 times the sample amount, and the eluent is petroleum ether: ethyl acetate = 4:1-1:1 gradient elution. After collecting the fraction with Rf value of 0.1-0.2 by TLC monitoring, the desired baicalin concentrate is obtained. (3) Add the baicalin enrichment obtained in step (2) to a polyol and stir to disperse it. Then add a stabilizer, continue stirring to homogenize, and then add another polyol. After heating and stirring to age, the baicalin liquid preparation is obtained.

2. The method for preparing the baicalin liquid formulation according to claim 1, characterized in that: In step (1), the mass ratio of water to coarse Scutellaria baicalensis root powder is 8-12:1, and the decoction time is 0.5-2h.

3. The method for preparing the baicalin liquid formulation according to claim 2, characterized in that: In step (1), the pH is adjusted to 1-2 for the first time, 6.5-7.5 for the second time, and 1-2 for the third time; the mass percentage of the ethanol solution is 40-60%.

4. The method for preparing the baicalin liquid formulation according to claim 3, characterized in that: In step (2), the hydrochloric acid solution has a mass percentage of 2-4%. Based on 1g of baicalin, 10-20mL of hydrochloric acid solution is added to each gram of baicalin.

5. The method for preparing the baicalin liquid formulation according to claim 4, characterized in that: In step (2), the temperature of the water bath reflux is 90-100℃ and the time is 0.5-2h; the pH of the solution is adjusted to 6-7.

6. The method for preparing the baicalin liquid formulation according to claim 5, characterized in that: In step (3), the polyol is selected from at least one of 1,3-propanediol, butanediol, methylpropanediol, glycerol and dipropylene glycol; the stabilizer is selected from at least one of Tween 20, Tween-80, lecithin, polyethylene glycol, AEO-3, AEO-7, AEO-9, poloxamer and ppg-5 cetyl ether-20.

7. The method for preparing the baicalin liquid formulation according to claim 6, characterized in that: In step (3), the total added mass of the polyol and stabilizer is ≥ 5% of the total mass of the system, and the mass ratio of the polyol and stabilizer is 1:4-3:

2.

8. The method for preparing the baicalin liquid formulation according to claim 6, characterized in that: In step (3), the polyol is added as follows: 40% is added first to disperse baicalein, and then the remaining 60% is added; the homogenization time is 2-3 hours and the temperature is 25-80℃; the aging time is ≥8 hours and the temperature is 20-80℃.

9. A baicalin liquid preparation prepared by the preparation method according to any one of claims 1-8.

10. The use of the baicalin liquid formulation according to claim 9 in cosmetics or daily chemical products.

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