A quercetin-loaded antibacterial oleogel and its preparation method

Abstract

This invention discloses a quercetin-loaded antibacterial oleogel and its preparation method. The oleogel preparation method includes: forming a homogeneous solution of quercetin dissolved in ethanol and vegetable oil under heating and stirring, ensuring complete dissolution of quercetin in the vegetable oil; then melting a gelling agent in the liquid vegetable oil and refrigerating overnight to obtain a semi-solid oleogel; and then further removing ethanol from the semi-solid oleogel by low-speed vacuum distillation to obtain the target oleogel. This invention utilizes ethanol as a co-solvent, increasing the solubility of quercetin in the oleogel; secondly, the addition of ethanol alters the crystal morphology of the oleogel, primarily forming β'-type crystals, and this crystal structure is retained even after ethanol removal. This structure effectively prevents quercetin aggregation and recrystallization, reduces the crystal particle size of quercetin, and further improves the bioavailability of quercetin. Antibacterial experiments showed that the antibacterial effect of ethanol-assisted quercetin-loaded oleogels was significantly improved compared to oleogel systems with or without quercetin. When the amount of quercetin added was approximately 0.94% of the liquid vegetable oil mass, the antibacterial rate reached nearly 100%. This invention utilizes ethanol as a co-solvent to uniformly disperse poorly oil-soluble bioactive substances in oleogels, providing an effective solution for constructing lipid products loaded with bioactive components.

Description

Technical Field

[0001] This invention belongs to the field of functional food and food processing technology, specifically relating to a quercetin-loaded antibacterial oleogel and its preparation method. Background Technology

[0002] Oil gels are complex microstructure systems that utilize thermally reversible three-dimensional gel networks constructed by gelling agents to restrict the flow of liquid oils, giving them solid properties. They represent a novel and safe plastic alternative to fats. Furthermore, oil gels can serve as delivery systems for bioactive ingredients, improving their stability, bioavailability, and targeted or controlled release, showing broad application prospects in food science and the pharmaceutical industry. Beeswax and β-sitosterol are food additives. Beeswax is a recognized gelling agent for edible oils, exhibiting high gelling efficiency even at very low crystallinity fractions. While stable oil gels formed by β-sitosterol alone exhibit some instability, when β-sitosterol is mixed with certain reagents such as beeswax in appropriate proportions, it can gelle a range of organic solvents by forming stable self-assembled networks. Moreover, oil gels resulting from mixed self-assembled structures formed when gelling agents are used in combination exhibit superior physical properties compared to oil gels formed from single-component self-assembled structures.

[0003] Quercetin is a flavonoid compound with rich medicinal value, including antioxidant, antibacterial, anti-inflammatory, and anticancer properties. Although quercetin offers numerous health benefits, its low stability, low water solubility (only 0.01 mg / mL), and low lipid solubility result in typically low bioavailability (<10%), significantly limiting its expected biological effects and practical applications. In recent years, the development of oleogels has provided a new strategy to improve the bioavailability of quercetin. Currently, many studies focus on oleogels as carriers of hydrophobic active substances, such as curcumin, vitamin E, and β-carotene. Experimental results show that encapsulating these active substances not only improves their stability and bioavailability but also significantly enhances the antioxidant and anti-inflammatory functions of the oleogel. However, to date, due to the low lipid solubility of quercetin, oleogel-based quercetin delivery systems remain relatively rare. Therefore, developing a quercetin-loaded oleogel system is of great significance for improving the bioavailability of quercetin and expanding the further applications of oleogel systems. Summary of the Invention

[0004] The purpose of this invention is to improve the bioavailability of quercetin by providing a quercetin-loaded antibacterial oleogel and its preparation method. The oleogel preparation method uses ethanol as a co-solvent, which not only increases the solubility of quercetin in the oleogel but also alters the crystal morphology of the oleogel, primarily producing β'-type crystals, which are retained even after the removal of ethanol. The resulting oleogel effectively prevents the aggregation and recrystallization of quercetin, reduces the crystal particle size of quercetin, further improves the bioavailability of quercetin, and provides a long-lasting and stable antibacterial effect.

[0005] To achieve the above objectives, the quercetin-loaded antibacterial oleogel provided by the present invention is prepared by the following method:

[0006] Step 1: Dissolve quercetin in anhydrous ethanol to obtain an alcoholic solution of quercetin;

[0007] Step 2: Disperse the alcoholic solution of quercetin evenly in liquid vegetable oil to obtain an oil solution of quercetin.

[0008] Step 3: Add the oleogel agent to the quercetin oil solution, heat to melt and obtain a viscous fluid, then refrigerate to obtain a semi-solid oleogel;

[0009] Step 4: The semi-solid olegel is subjected to vacuum distillation to completely remove ethanol, resulting in quercetin-loaded antibacterial olegel.

[0010] Furthermore, in step 1 above, the mass concentration of quercetin in anhydrous ethanol is 10–80 mg / mL.

[0011] Furthermore, in step 2 above, the liquid vegetable oil is selected from at least one of sea buckthorn fruit oil, tea oil, walnut oil, peony seed oil, sunflower seed oil, and corn oil, and preferably the amount of quercetin added is 0.3% to 1.3% of the mass of the liquid vegetable oil.

[0012] Furthermore, in step 3 above, the oleogel is one or both of beeswax and β-sitosterol. Beeswax can form a gel at a low concentration, and β-sitosterol has the effect of modifying crystal form. The combination of the two is beneficial for forming a three-dimensional network structure. Preferably, the amount of oleogel added is 15% to 25% of the mass of the liquid vegetable oil.

[0013] Furthermore, in step 3 above, the heating and melting temperature is 70–90 °C.

[0014] Furthermore, in step 3 above, the refrigeration temperature is 0–4 °C and the time is 12–24 h.

[0015] Furthermore, in step 4 above, the temperature of the vacuum distillation is 30–40 °C, the vacuum degree is 800–1000 Pa, and the time is 0.5–4 h.

[0016] The beneficial effects of this invention are as follows:

[0017] This invention uses anhydrous ethanol as a co-solvent for quercetin. Quercetin dissolved in ethanol is mixed with liquid vegetable oil under heating and stirring to form a homogeneous solution, ensuring complete dissolution of quercetin in the liquid vegetable oil. A gelling agent is then melted into the liquid vegetable oil and refrigerated overnight to obtain a semi-solid oleogel. Finally, the semi-solid oleogel is subjected to low-speed vacuum distillation of ethanol to obtain an antibacterial oleogel. In this invention, the co-solubilizing effect of ethanol allows the poorly lipid-soluble quercetin to dissolve in the liquid vegetable oil and be uniformly embedded in the oleogel crystals. This not only increases the quercetin loading but also alters the crystallization form of the oleogel, resulting in a predominantly β'-type crystal with a smoother, softer texture and better spreadability. The high loading capacity and excellent spreadability of quercetin endow the oleogel with better antibacterial properties, increasing the antibacterial effect by more than 40% compared to oleogels with or without quercetin alone. When the amount of quercetin added is approximately 0.94% of the liquid vegetable oil mass, the antibacterial rate can approach 100%. This invention pioneers a new method for uniformly dispersing poorly lipid-soluble bioactive substances in oleogels, providing an effective solution for constructing lipid products loaded with functional bioactive ingredients. Attached Figure Description

[0018] Figure 1 These are Fourier transform infrared schematic diagrams of the oleogels in Examples 1-6 and Comparative Examples 1-4.

[0019] Figure 2 These are polarized microscope schematic diagrams of the oleogels in Examples 1-6 and Comparative Examples 1-4.

[0020] Figure 3 These are XRD schematic diagrams of the oleogels of Examples 1-6 and Comparative Examples 2-4.

[0021] Figure 4 These are schematic diagrams illustrating the antibacterial properties of the oleogels in Examples 1-6 and Comparative Examples 2-4. Detailed Implementation

[0022] The present invention will be further described in detail below with reference to the accompanying drawings and embodiments, but the scope of protection of the present invention is not limited to these embodiments.

[0023] Example 1

[0024] Step 1: Dissolve 12.5 mg of quercetin in 1 mL of anhydrous ethanol to obtain an alcoholic solution of quercetin.

[0025] Step 2: Add 4 g of sea buckthorn fruit oil to the quercetin alcohol solution from Step 1, heat and stir at 90 °C to dissolve the quercetin alcohol solution in the sea buckthorn fruit oil, and obtain an oil solution of quercetin.

[0026] Step 3: Add 0.53 g beeswax and 0.80 g β-sitosterol to the quercetin oil solution from Step 2, stir at 600 rpm for 30 min at 90℃ to obtain a viscous fluid, cool to room temperature and then refrigerate at 4℃ for 24 h to obtain a semi-solid oleogel.

[0027] Step 4: The semi-solid oleogel was distilled under reduced pressure at 35 °C for 1 h using a rotary evaporator to completely remove ethanol, resulting in a quercetin-loaded antibacterial oleogel.

[0028] Example 2

[0029] In step 1 of this embodiment, 25 mg of quercetin was dissolved in 1 mL of anhydrous ethanol to obtain an alcoholic solution of quercetin. The other steps were the same as in Example 1 to obtain a quercetin-loaded antibacterial oleogel.

[0030] Example 3

[0031] In step 1 of this embodiment, 37.5 mg of quercetin was dissolved in 1 mL of anhydrous ethanol to obtain an alcoholic solution of quercetin. The other steps were the same as in Example 1 to obtain a quercetin-loaded antibacterial oleogel.

[0032] Example 4

[0033] In step 1 of this embodiment, 50 mg of quercetin is dissolved in 1 mL of anhydrous ethanol to obtain an alcoholic solution of quercetin. The other steps are the same as in Example 1 to obtain a quercetin-loaded antibacterial oleogel.

[0034] Example 5

[0035] In step 3 of this embodiment, 0.28 g of beeswax and 0.42 g of β-sitosterol were added to the quercetin oil solution from step 2. The mixture was stirred at 600 rpm for 30 min at 90 °C to obtain a viscous fluid. After cooling to room temperature, the fluid was refrigerated at 4 °C for 24 h to obtain a semi-solid oleogel. The other steps were the same as in Example 4 to obtain a quercetin-loaded antibacterial oleogel.

[0036] Example 6

[0037] In step 3 of this embodiment, 0.40 g of beeswax and 0.60 g of β-sitosterol were added to the quercetin oil solution from step 2. The mixture was stirred at 600 rpm for 30 min at 90 °C to obtain a viscous fluid. After cooling to room temperature, the fluid was refrigerated at 4 °C for 24 h to obtain a semi-solid oleogel. The other steps were the same as in Example 4 to obtain a quercetin-loaded antibacterial oleogel.

[0038] Comparative Example 1

[0039] 50 mg of quercetin was added to 4 g of sea buckthorn fruit oil, and homogenized by ultrasound to ensure that the quercetin was evenly dispersed in the sea buckthorn fruit oil, thus obtaining an oil solution of quercetin.

[0040] Comparative Example 2

[0041] Weigh 4 g of sea buckthorn fruit oil, then add 0.53 g of beeswax and 0.80 g of β-sitosterol, stir at 600 rpm for 30 min at 90 ℃, cool at room temperature and refrigerate at 4 ℃ for 24 h to obtain oil gel.

[0042] Comparative Example 3

[0043] Add 1 mL of anhydrous ethanol to 4 g of sea buckthorn fruit oil, then add 0.53 g of beeswax and 0.80 g of β-sitosterol. Stir at 600 rpm for 30 min at 90 ℃, cool to room temperature, and then refrigerate at 4 ℃ for 24 h to obtain an oil gel.

[0044] Comparative Example 4

[0045] 50 mg of quercetin was ultrasonically dispersed in 4 g of sea buckthorn fruit oil, then 0.53 g of beeswax and 0.80 g of β-sitosterol were added. The mixture was stirred at 600 rpm for 30 min at 90 ℃, cooled to room temperature, and then refrigerated at 4 ℃ for 24 h to obtain an oil gel.

[0046] The cross-linking and quercetin encapsulation within the oleogels of Examples 1-6 and Comparative Examples 1-4 were detected using attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR) in the wavelength range of 400-4000 cm⁻¹. -1 The result is as follows Figure 1 As shown, all oleogels are in the range of 3000–2700 cm. -1 1745 cm -1 and 1460 cm -1 1300~1100 cm -1 And 721 cm -1 Similar characteristic absorption peaks were observed at all locations. Unlike other systems, in Comparative Example 3 containing ethanol, the peaks were observed at 3600–3100 cm⁻¹. -1 and 880 cm -1 Two characteristic peaks belonging to the formation of hydrogen bonds between ethanol molecules were also observed nearby. These two characteristic peaks were not observed in Examples 1-6, where ethanol was removed by vacuum distillation, demonstrating that low-speed vacuum distillation can effectively remove ethanol from the oleogel.

[0047] Figure 2The results are presented using polarized light microscopy (PLM) to evaluate the oleogels of Comparative Examples 1-4 and Examples 1-6. In Comparative Example 2, the pure oleogel exhibited large crystal particle sizes, with aggregated crystals and a dense network structure. In Comparative Example 3, after the addition of ethanol, the oleogel showed smaller, finer crystal particles, significantly smaller crystal aggregates, and more pronounced gaps between crystals, indicating that the addition of ethanol facilitated the formation of a new crystal network structure. Furthermore, this crystal structure was effectively preserved even after ethanol removal via low-speed vacuum distillation, and the crystal network structure of the oleogels in Examples 1-6 was essentially consistent with that of Comparative Example 3. Additionally, in Comparative Examples 1 and 4, distinct yellow rod-shaped crystals of quercetin were observed, indicating that quercetin was not fully dissolved in pure oil and pure oleogel and existed primarily as large-particle crystals. In Examples 1-6, quercetin was mainly distributed uniformly in the oleogel crystal structure as small particles, which confirms that the addition of ethanol can not only change the crystal network structure of the oleogel, but also further improve the solubility of quercetin in the oleogel, thus helping to improve its bioavailability.

[0048] Figure 3 The X-ray diffraction (XRD) patterns of the oleogels from Comparative Examples 2–4 and Examples 1–6 are shown. The diffraction peaks of each group of oleogels mainly consist of characteristic peaks of the β-type and β′-type crystals. β-type crystals have larger and more compact grains, with characteristic peaks mainly appearing in the 4.5–4.6 Å region; β′-type crystals have smaller and finer grains, exhibiting good plasticity and spreadability, with characteristic peaks mainly appearing near 3.8 Å and 4.2 Å. Comparative analysis of the XRD results from Comparative Example 2 and Comparative Example 3 reveals that the addition of ethanol significantly broadens and reduces the size of the diffraction characteristic peaks belonging to the β-type crystals in the oleogel structure, making the oleogel structure predominantly β′-type. Furthermore, this crystal structure is effectively preserved even after ethanol removal by low-speed vacuum distillation. The XRD patterns of the oleogels from Examples 1–6 are basically similar to those of Comparative Example 3. This result is consistent with the results of polarized light microscopy, confirming that the addition of ethanol can change the crystal network structure of olegels, reduce the fixed β-type crystals in olegels, and make the olegels mainly composed of small, fine, highly plastic, and easily spreadable β′-type crystals, effectively expanding the applicability of olegel systems.

[0049] The in vitro antibacterial activity of the oleogels prepared in Examples 1-6 and Comparative Examples 2-4 was further evaluated: oleogels with a concentration of 10 were prepared respectively. 8CFU / mL of *Escherichia coli*, *Staphylococcus aureus*, and *Candida albicans* bacterial suspensions were prepared and diluted. 100 μL of each suspension was evenly spread onto a solid culture medium and allowed to stand for 10 min to allow complete adhesion. Then, 0.2 g of oleogloss was evenly dispersed onto the medium using a spreader. The medium was incubated at 37 ℃ for 12–18 h, and the colony status was observed and recorded. Each sample was tested three times.

[0050] like Figure 4 As shown, compared with oleogels with or without quercetin (Comparative Examples 2-4), the quercetin-loaded oleogels prepared in Examples 1-6 exhibited significant antibacterial effects against all three bacteria. In Example 3, when the amount of quercetin added was approximately 0.94% of the mass of the liquid vegetable oil, the antibacterial rate of the oleogels approached 100%. Therefore, the preparation method of this invention, by introducing ethanol as a co-solvent, not only increases the solubility of quercetin in the oleogels but also ensures that the crystal structure of the oleogels is mainly composed of β′-type crystals with good spreadability, further improving the bioavailability of quercetin and giving the quercetin-loaded oleogels excellent antibacterial properties.

Claims

1. A method of preparing a quercetin loaded bacteriostatic oil gel, characterized by: The preparation method comprises the following steps: Step 1: dissolving quercetin in anhydrous ethanol to obtain an alcohol solution of quercetin; Step 2: uniformly dispersing the alcohol solution of quercetin in liquid vegetable oil to obtain an oil solution of quercetin; the liquid vegetable oil is selected from at least one of sea buckthorn fruit oil, tea oil, walnut oil, peony seed oil, sunflower seed oil and corn oil; Step 3: adding an oil gel agent to the oil solution of quercetin, melting by heating to obtain a viscous fluid, and refrigerating to obtain a semi-solid oil gel; the oil gel agent is one or both of beeswax and beta-sitosterol; Step 4: performing vacuum distillation on the semi-solid oil gel to completely remove ethanol to obtain a quercetin-loaded antibacterial oil gel.

2. The method of claim 1, wherein the quercetin-antibacterial oil gel is prepared by the following steps: In step 1, the mass concentration of quercetin in anhydrous ethanol is 10-80 mg / mL.

3. The method of claim 1, wherein the quercetin-antibacterial oil gel is prepared by the following steps: In step 2, the addition amount of quercetin is 0.3%-1.3% of the mass of the liquid vegetable oil.

4. The method of claim 1, wherein the quercetin-antibacterial oil gel is prepared by the following steps: In step 3, the addition amount of the oil gel agent is 15%-25% of the mass of the liquid vegetable oil.

5. The method of claim 1, wherein the quercetin-antibacterial oil gel is prepared by the following steps: In step 3, the temperature of the heating and melting is 70-90 ℃.

6. The method of claim 1, wherein the quercetin-antibacterial oil gel is prepared by the following steps: In step 3, the refrigeration temperature is 0-4 ℃, and the refrigeration time is 12-24 h.

7. The method of claim 1, wherein the quercetin-antibacterial oil gel is prepared by the following steps: In step 4, the temperature of the vacuum distillation is 30-40 ℃, the vacuum degree is 800-1000 Pa, and the time is 0.5-4 h.

8. A quercetin-loaded antibacterial oil gel prepared by the preparation method of any one of claims 1-7.

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