Preparation method and application of borage eucalyptus leaf oil microemulsion gel

Abstract

The application belongs to the technical field of biological medicine, and discloses a preparation method and application of a bunge leaf oil microemulsion gel. The method is characterized in that: 5-O-beta-D-glucosyl shikonin is generated by fermentation of Streptomyces CGMCC 4.1001 to modify shikonin, and then the 5-O-beta-D-glucosyl shikonin is combined with eucalyptus oil, a composite surfactant (Tween 80 / Span 80 = 4:1), n-butanol and water to construct an O / W type microemulsion, then a xanthan gum / kaolin gum mixture is introduced and calcium ions are used to trigger in-situ crosslinking to form a nano-network microemulsion gel. The application successfully solves multiple technical contradictions such as poor water solubility of shikonin, easy evaporation of eucalyptus oil, poor compatibility of microemulsion and gel, etc. by using a three-in-one technical path of biotransformation modification-microemulsion construction-in-situ gelation, and realizes the unity of high drug loading, high stability, high transdermal and good use feeling in a single delivery system. The application is suitable for external treatment of skin inflammation, wound healing and itching relief with moderate pH and no irritation.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology and relates to a method for preparing microemulsion gel of eucalyptus leaf oil and its application. Background Technology

[0002] In the field of modern topical medicine and functional skin care formulations, natural plant active ingredients have attracted much attention due to their excellent biocompatibility and multiple pharmacological activities. Shikonin and its derivatives contained in comfrey have significant anti-inflammatory, antibacterial, wound-healing, and antioxidant effects, and have been widely used as adjunctive treatments for skin diseases such as burns, eczema, and dermatitis. Eucalyptus oil, with its excellent volatile aromatic properties, broad-spectrum antibacterial ability, and local cooling sensation, occupies an important position in respiratory preparations and topical anti-inflammatory products.

[0003] Shikonin, a naphthoquinone compound, has a highly hydrophobic molecular structure and is almost insoluble in water. This makes it difficult to disperse uniformly in conventional aqueous gel or emulsion systems, limiting not only the effective drug loading capacity but also severely restricting its transdermal penetration efficiency and targeted delivery capability. Eucalyptus oil is mainly composed of volatile monoterpenoids such as 1,8-cineole, which are easily lost through volatilization at room temperature and are sensitive to light, heat, and oxygen. The active ingredients exhibit poor stability during formulation storage and use, making it difficult to maintain long-lasting efficacy. These physicochemical properties combined result in significantly low bioavailability when used alone or in combination. Even with conventional solubilization methods (such as adding surfactants or organic cosolvents), it is difficult to balance stability, safety, and transdermal efficiency, and may even cause skin irritation or damage to the skin barrier function.

[0004] While existing technologies attempt to improve the solubility of shikonin through microemulsion systems and utilize oil phase encapsulation to suppress the volatilization of eucalyptus oil, microemulsions themselves are thermodynamically stable but kinetically fragile isotropic systems. Their low viscosity characteristics make it difficult to meet the requirements of topical gels for retention and sustained release. If microemulsions are directly physically mixed with traditional gel matrices (such as carbomer), phase separation or demulsification is easily triggered by changes in ionic strength, pH, or interfacial tension, leading to the burst release or precipitation of active ingredients, which exacerbates stability problems. Even when using polymer thickening or cross-linking strategies to improve the viscoelasticity of the system, the original nanoscale particle size advantage of the microemulsion is often sacrificed, weakening its ability to promote transdermal absorption. Summary of the Invention

[0005] To achieve the above-mentioned objectives, this invention provides a method for preparing eucalyptus leaf oil microemulsion gel and its application. The method introduces a shikonin derivative modified by specific microbial fermentation as the core active ingredient, and together with eucalyptus leaf oil, constructs a thermodynamically stable O / W microemulsion system. Then, through in-situ crosslinking, a microemulsion gel with a nano-network structure is formed. This achieves efficient solubilization of hydrophobic shikonin derivative, physical locking of volatile eucalyptus leaf oil, and synergistic optimization of the overall rheological properties and long-term stability of the system in a single delivery system.

[0006] The preparation method of the eucalyptus leaf oil microemulsion gel of the present invention includes the following steps: First, the eucalyptus root powder is pretreated and inoculated with a specific strain for solid-state fermentation to obtain a structurally modified shikonin derivative; second, the obtained shikonin derivative is mixed with eucalyptus leaf oil, a composite surfactant, a co-surfactant, and deionized water in a specific ratio, and homogenized and emulsified to form a transparent or semi-transparent O / W type microemulsion; finally, a natural polysaccharide gel matrix precursor is introduced into the microemulsion system, and the in-situ gelation reaction is triggered by adjusting the pH value or adding crosslinking ions to form a microemulsion gel with a three-dimensional nano-network structure.

[0007] In the first step, the particle size of the *Lithospermum erythrorhizon* root powder is 80-120 mesh, and the moisture content is controlled at 8%-12%. The specific strain is a *Streptomyces* strain obtained through screening, with the preservation number CGMCC 4.1001. This strain secretes β-glucosyltransferase during solid-state fermentation, which can selectively glycosylate the C-5 hydroxyl group of the shikonin molecule to generate 5-O-β-D-glucosylshikonin. The solid-state fermentation culture medium consists of *Lithospermum erythrorhizon* root powder, wheat bran, ammonium sulfate, potassium dihydrogen phosphate, and trace element solution, wherein the mass ratio of *Lithospermum erythrorhizon* root powder to wheat bran is 3:1, the fermentation temperature is 28℃, the relative humidity is 75%, and the fermentation period is 96 hours. After fermentation, the fermentation product was extracted with a 70% ethanol aqueous solution at a temperature of 50°C for 2 hours at a material-to-liquid ratio of 1:10 (g / mL). The extract was concentrated under reduced pressure, purified with macroporous resin, and freeze-dried to obtain shikonin derivative powder with a 5-O-β-D-glucosylshikonin content higher than 85%.

[0008] In the second step, the microemulsion system comprises: 0.5%-2.0% (mass fraction, the same below) of shikonin derivative, 1.0%-5.0% of eucalyptus oil, 8.0%-15.0% of composite surfactant, 2.0%-6.0% of co-surfactant, and the balance being deionized water. The composite surfactant is a mixture of Tween 80 and Span 80 in a mass ratio of 4:1, with an HLB value of 12.8. The co-surfactant is n-butanol. The microemulsion is prepared using the phase transition temperature method: first, the shikonin derivative is completely dissolved in eucalyptus oil to form an oil phase; the composite surfactant and co-surfactant are mixed evenly to form the surfactant phase; the oil phase is slowly added to the surfactant phase and stirred until a clear and transparent isotropic system is formed; then, deionized water is added dropwise under a 40°C water bath while continuously stirring at 800 rpm until the system spontaneously forms a transparent microemulsion.

[0009] In the third step, the natural polysaccharide gel matrix precursor is a mixture of xanthan gum and gellan gum in a mass ratio of 3:2, with a total addition amount of 0.8%-1.5% of the total mass of the microemulsion system. The crosslinking ion is calcium ion, derived from calcium chloride solution, with a concentration of 0.1 mol / L, and an addition amount of 0.5%-1.0% of the total mass of the microemulsion system. The in-situ gelation process is as follows: Xanthan gum and gellan gum are pre-dispersed in a portion of deionized water, swollen for 30 minutes, heated to 85°C to completely dissolve, cooled to 40°C, and added to the prepared microemulsion system, stirred evenly; then, under constant temperature of 40°C, calcium chloride solution is added dropwise at a rate of 1 mL / min while maintaining a stirring rate of 300 rpm. After the addition is complete, stirring continues for 10 minutes, and the mixture is allowed to stand and cool to room temperature to obtain the eucalyptus leaf oil microemulsion gel. The microemulsion gel has an apparent viscosity of 8000-15000 mPa·s at 25°C, and its storage modulus G' is greater than its loss modulus G'', indicating that it has typical gel rheological properties.

[0010] In a preferred embodiment of the present invention, the eucalyptus oil has a 1,8-cineole content higher than 70% and a density of 0.910-0.930 g / cm³. 3 The refractive index is 1.458-1.468.

[0011] In another preferred embodiment of the present invention, the microemulsion gel may further contain a transdermal permeability enhancer, wherein the transdermal permeability enhancer is azone, and the amount added is 0.5%-1.0% of the total mass of the microemulsion system. The azone is added to the oil phase before microemulsion formation and is dissolved together with the shikonin derivative and eucalyptus oil.

[0012] The application of the eucalyptus leaf oil microemulsion gel described in this invention specifically refers to its use as a topical preparation in treating skin inflammation, promoting wound healing, or relieving skin itching. The topical preparation is in gel form and is applied directly to the affected area 1-2 times daily. The microemulsion gel has a pH of 5.5-6.5, which is close to the physiological pH of the skin and is non-irritating.

[0013] Compared with the prior art, the beneficial effects of the present invention are:

[0014] 1. In the preparation method of the microemulsion gel described in this invention, the microbial fermentation modification step is a fundamental technical means to solve the problem of poor water solubility of shikonin. Traditional physical solubilization methods only achieve solubilization through surfactant micelle encapsulation, which has defects such as low drug loading and easy precipitation due to dilution. However, this invention introduces hydrophilic glucose groups into the shikonin molecule through glycosylation modification mediated by Streptomyces CGMCC 4.1001, thereby improving its water solubility and chemical stability at the molecular structure level. This allows it to be well miscible with eucalyptus oil inside the microemulsion oil droplets, and at the same time, the hydrophilicity of the glycosyl groups enhances its compatibility with the aqueous phase at the microemulsion interface. Thus, it significantly improves the drug loading and system stability without increasing the amount of surfactant.

[0015] 2. The in-situ crosslinking strategy of the microemulsion gel described in this invention solves the technical problem of poor compatibility between microemulsions and gel matrices. Traditional methods physically mix pre-formed gels with microemulsions, which easily leads to the destruction of the microemulsion nanostructure due to polymer chain entanglement. This invention employs a synergistic gel system of xanthan gum and gellan gum. A gel precursor is introduced after microemulsion formation, and calcium ions trigger the formation of the double helix structure of gellan gum. Xanthan gum is then embedded into this network through non-covalent interactions, forming a three-dimensional network structure with nanopores. The pore size of this network is much larger than the microemulsion particle size (25-45 nm), thus the microemulsion particles are completely embedded in the gel network. This maintains the nanoscale properties of the microemulsion to promote transdermal absorption and provides the system with sufficient mechanical strength to prevent flow and the escape of volatile components.

[0016] 3. The composite surfactant system described in this invention is designed to balance microemulsion forming ability and skin safety. The combination of Tween 80 and Span 80 not only provides a suitable HLB value to stabilize O / W microemulsions, but both are also pharmaceutical excipients with low toxicity. The co-surfactant n-butanol has a moderate carbon chain length, which can reduce interfacial tension and promote spontaneous microemulsion formation, and its low volatility ensures that its residue in the final gel is controllable, avoiding skin irritation.

[0017] 4. The eucalyptus leaf oil microemulsion gel of this invention requires no organic solvents during preparation, and all components are pharmaceutical-grade or food-grade raw materials, meeting the safety requirements for topical preparations. The microemulsion gel is translucent, uniform in color, without a grainy feel, has good spreadability, forms a film quickly after application, and has no greasy feel, providing a superior user experience compared to traditional creams or ointments. Detailed Implementation

[0018] This invention provides a method for preparing eucalyptus leaf oil microemulsion gel and its application. The core of the method lies in modifying the structure of shikonin through microbial fermentation to improve its water solubility, and then co-constructing a thermodynamically stable O / W microemulsion system with eucalyptus leaf oil. On this basis, a microemulsion gel with a nano-network structure is formed through in-situ cross-linking. This allows for the simultaneous and efficient solubilization of hydrophobic active ingredients, physical locking of volatile components, and synergistic optimization of the system's rheological properties and long-term stability in a single delivery system.

[0019] The technical solution of the present invention will be described in detail below with reference to specific embodiments and comparative examples, so as to ensure that those skilled in the art can fully understand and implement the present invention.

[0020] Example 1: Shikonin derivative 1.2%; Eucalyptus oil 3.0%; Compound surfactant 12.0%; Co-surfactant 4.0%; Gel matrix 1.2% (xanthan gum: gellan gum = 3:2); Calcium chloride 0.8%; No transdermal penetration enhancer; pH 6.0;

[0021] Preparation process: Shikonin biomodification → microemulsion preparation → gel matrix dispersion → in-situ crosslinking of calcium ions → finished product.

[0022] Example 2: 0.5% of shikonin derivative, the rest of the formulation and process are the same as in Example 1;

[0023] Preparation process: Same as in Example 1 (with adjustment of active ingredient ratio).

[0024] Example 3: 2.0% of shikonin derivative, the rest of the formulation and process are the same as in Example 1;

[0025] Preparation process: Same as in Example 1 (with adjustment of active ingredient ratio).

[0026] Example 4: Gel matrix 0.8%, other formulations and processes are the same as in Example 1;

[0027] Preparation process: Same as in Example 1 (adjustment of gel matrix amount).

[0028] Example 5: Gel matrix 1.5%, other formulations and processes are the same as in Example 1;

[0029] Preparation process: Same as in Example 1 (adjustment of gel matrix amount).

[0030] Example 6: Add 0.8% azone, the rest of the formulation and process are the same as in Example 1;

[0031] Preparation process: Same as in Example 1 (with the addition of transdermal transdermal accelerator).

[0032] Example 7: Eucalyptus oil 1.0%, the rest of the formula and process are the same as in Example 1;

[0033] Preparation process: Same as Example 1 (with adjusted eucalyptus oil ratio)

[0034] Example 8: Eucalyptus oil 5.0%, the rest of the formula and process are the same as in Example 1.

[0035] Preparation process: Same as in Example 1 (with adjustment of eucalyptus oil ratio).

[0036] Comparative Example 1: Unmodified shikonin was used to replace the biomodified derivative, and the rest of the formulation and process were the same as in Example 1;

[0037] Preparation process: Direct dissolution of shikonin → microemulsion preparation → gelation → finished product.

[0038] Comparative Example 2: No biomodification or microemulsion step was performed; shikonin and eucalyptus oil were directly mixed into xanthan gum gel, and the rest of the process was the same as in Example 1.

[0039] Preparation process: mixing of active ingredients → dispersion of gel matrix → finished product.

[0040] Test method:

[0041] Stability and drug loading tests: The retention rate of active ingredients was tested after 6 months of accelerated storage; the maximum drug loading was determined; and the appearance and phase stability were observed.

[0042] Transdermal performance testing: The cumulative transdermal dose over 24 hours was determined using the Franz diffusion cell method; skin irritation was assessed using a patch test.

[0043] Physicochemical performance testing: microemulsion particle size was measured by dynamic light scattering method; gel viscosity was measured by rotational rheometer; and the pH of the system was measured by pH meter.

[0044] The test data comparisons are shown in Table 1 and Table 2.

[0045] Table 1. Comparison of drug loading, microemulsion particle size, viscosity at 25℃, and skin irritation.

[0046] Test Project Drug loading (%) Microemulsion particle size (nm) Viscosity at 25°C (mPa・s) Skin irritation Example 1 4.2 35 12000 none Example 2 3.5 32 10000 none Example 3 5 40 14000 none Example 4 4.1 34 8500 none Example 5 4.3 36 14500 none Example 6 4.2 33 11500 none Example 7 2.5 30 9500 none Example 8 7 42 13500 none Comparative Example 1 1.8 65 8000 slight Comparative Example 2 2.2 - 9000 none

[0047] Table 2. Comparison of 6-month activity retention rate, transdermal absorption of shikonin, and transdermal absorption of eucalyptol.

[0048] Test Project 6-month activity retention rate (%) <![CDATA[The percutaneous amount of shikonin (μg / cm 2 )]]> <![CDATA[Eucalyptol transdermal amount (μg / cm 2 )]]> Example 1 93 88 135 Example 2 95 85 130 Example 3 92 105 145 Example 4 90 92 140 Example 5 94 86 132 Example 6 92 108 148 Example 7 96 87 122 Example 8 91 95 150 Comparative Example 1 65 35 100 Comparative Example 2 58 28 85

[0049] Examples 1-8: Drug loading ≥2.5%, transdermal delivery ≥85 μg / cm³ 2 The results were far superior to those of the comparative example. Comparative example 1 had poor water solubility of shikonin due to the lack of biomodification, while comparative example 2 had insufficient stability and transdermal properties due to the traditional mixing without nano-solubilization. This demonstrates that biomodification + microemulsion + in-situ gel is the key to efficient delivery.

[0050] The proportion of biomodified shikonin was increased (Examples 2→1→3), and the drug loading and transdermal capacity were increased simultaneously; the gel matrix was increased (Examples 4→1→5), and the stability was enhanced, but the transdermal capacity decreased slightly; the addition of a transdermal activator (Example 6) further optimized the transdermal efficiency.

[0051] The embodiment features high retention of active ingredients, suitable for long-term storage; high drug loading capacity, reducing the frequency of medication; pH is compatible with the skin's physiological environment, causing no irritation; the preparation process is mild, suitable for industrial production; and it is applicable to scenarios such as skin inflammation and wound healing.

[0052] Compared to the unmodified (Comparative Example 1), the drug loading of the example was increased by 133% and the transdermal delivery was increased by 151%; compared to the traditional mixed gel (Comparative Example 2), the activity retention rate was increased by 57% and the transdermal delivery was increased by 211%, solving the industry problems of poor water solubility of shikonin and easy volatility of eucalyptus oil.

[0053] In summary, the method described in this invention, through the synergistic effect of biomodification and nanodelivery, can achieve the preparation of highly stable, highly drug-loaded, and highly transdermal eucalyptus leaf oil microemulsion gels with different parameter combinations, making it suitable for topical skin care and therapeutic preparations.

[0054] The above are merely preferred embodiments of the present invention and are 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 process for the preparation of a microemulsion gel of Eucalyptus globulus leaf oil characterized in that, Includes the following steps: (1) After pretreatment of the root powder of Lithospermum erythrorhizon, it was inoculated with Streptomyces strain for solid-state fermentation to obtain glycosylated Lithospermum erythrorhizon derivatives. The solid fermentation medium consists of Lithospermum root powder, wheat bran, ammonium sulfate, potassium dihydrogen phosphate, and trace element solution; The fermentation product was extracted with 70% ethanol, concentrated under reduced pressure, purified with macroporous resin and freeze-dried to obtain a shikonin derivative powder with a 5-O-β-D-glucosylshikonin content of more than 85%. (2) The shikonin derivative is mixed with eucalyptus oil, a composite surfactant, a co-surfactant and deionized water, and an O / W microemulsion is formed by phase transition temperature method. (3) A mixture of xanthan gum and gellan gum is added to the microemulsion system as a gel matrix precursor, and calcium chloride solution is added dropwise to introduce calcium ions to trigger in-situ crosslinking, forming a microemulsion gel with a three-dimensional nano-network structure.

2. The process for the preparation of a micellar gel of Eucalyptus oil of Alkanna tinctoria as claimed in claim 1, wherein, The particle size of the purple gromwell root powder is 80-120 mesh, and the moisture content is 8%-12%.

3. The process for the preparation of the Eucalyptus oil microemulsion gel of the plant Erythroxylum coca according to claim 1, characterized in that, The mass ratio of the purple gromwell root powder to wheat bran is 3:

1.

4. The process for the preparation of the Eucalyptus oil microemulsion gel of the Licorice plant as claimed in claim 1, wherein, The solid-state fermentation was carried out at a temperature of 28°C and a relative humidity of 75%, with a fermentation cycle of 96 hours.

5. The method for preparing the eucalyptus leaf oil microemulsion gel according to claim 1, characterized in that, The microemulsion system contains 0.5%-2.0% shikonin derivatives, 1.0%-5.0% eucalyptus oil, 8.0%-15.0% composite surfactant, 2.0%-6.0% co-surfactant, and the remainder is deionized water.

6. The method for preparing the eucalyptus leaf oil microemulsion gel according to claim 1, characterized in that, The composite surfactant is prepared by compounding Tween 80 and Span 80 in a mass ratio of 4:1, with an HLB value of 12.

8.

7. The method for preparing the eucalyptus leaf oil microemulsion gel according to claim 1, characterized in that, The co-surfactant is n-butanol.

8. The method for preparing the eucalyptus leaf oil microemulsion gel according to claim 1, characterized in that, The xanthan gum to gellan gum mass ratio is 3:2, and the total amount added is 0.8%-1.5% of the total mass of the microemulsion system.

9. The method for preparing the microemulsion gel of Eucalyptus leaf oil according to claim 1, characterized in that, The calcium chloride solution has a concentration of 0.1 mol / L and is added at a rate of 0.5%-1.0% of the total mass of the microemulsion system.

10. The method for preparing the eucalyptus leaf oil microemulsion gel according to claim 1, characterized in that, It also includes adding the transdermal penetration enhancer azone to the oil phase, with the addition amount being 0.5%-1.0% of the total mass of the microemulsion system.