Preparation method of luteolin liposome as well as product and application of luteolin liposome

Abstract

The invention provides a preparation method of luteolin liposome as well as a product and application of the luteolin liposome, and relates to the technical field of liposome. The preparation method of the luteolin liposome comprises the following steps: S1, dissolving lecithin, cholesterol and luteolin in absolute ethyl alcohol according to a mass ratio of (40-60): 15: (4-6), and carrying out reduced pressure spin-drying to form a film; and S2, hydration: mixing the film with a phosphate buffer solution or water with the pH value of 6.8-7.2, stirring and hydrating at 50-60 DEG C for 6-24 hours to form a suspension, and performing probe ultrasonic treatment on the suspension. The obtained luteolin liposome has the advantages of high encapsulation efficiency, low particle size, high stability and good slow release effect. When being applied to cosmetics which can relieve skin and have no irritation effect on sensitive skin, the composition has a remarkable slow release effect.

Description

Technical Field

[0001] The invention belongs to the technical field of liposomes, and specifically relates to a preparation method of luteolin liposomes and a product and application thereof. Background Art

[0002] Luteolin is a flavonoid compound with the following chemical structure:

[0003]

[0004] Luteolin has anti-inflammatory, anti-allergic, uric acid-lowering, antibacterial and antiviral pharmacological activities. Luteolin is added to cosmetics to soothe, inhibit tyrosinase activity, and inhibit oxidative free radicals. However, since luteolin is a polyphenolic hydroxyl compound, the intermolecular force and lattice energy between its hydroxyl groups are high, and its lipophilicity and hydrophilicity are poor. Its special structure leads to low solubility in a variety of solvents. Therefore, in the application of cosmetics, luteolin has the problems of poor stability, low solubility, and low bioavailability.

[0005] Jiang Yaling (Jiang Yaling, Li Wenyuan, Feng Shuang, et al. Research progress on the structural modification and biological activity of luteolin [J]. Chinese Herbal Medicine, 2023, 54(20): 6889-6902) et al. modified the structure of luteolin to enhance its biological activity. They modified the carbonyl group on the benzene ring of luteolin by esterification, etherification, substitution, coordination, etc., optimized the structure of luteolin, enhanced its solubility, and improved its bioavailability. However, the chemical synthesis method is inevitably limited, and the cost, operability, and yield still need to be improved.

[0006] Liposomes, also known as lipid globules or liquid crystal microcapsules, are microvesicles formed by encapsulating active substances in a lipid bilayer. The phospholipid bilayer is composed of a highly hydrophilic head and a hydrophobic tail. The outer and inner surfaces of the liposome are tightly arranged hydrophilic heads, and the hydrophobic tail is in the middle of the membrane. The liposome structure is highly similar to biological membranes, with good biocompatibility and biodegradability. Studies have shown that the use of liposome encapsulation technology for active substances and the application of its products in cosmetics can accelerate the entry into the deep layers of the skin. After the encapsulated active substances are absorbed through the skin, more active substances can remain between the epidermis and the dermis, and the stability of unstable active substances can also be enhanced.

[0007] Therefore, the use of liposome encapsulation technology to prepare luteolin liposomes is expected to solve the problems of poor water solubility, rapid metabolism, short half-life and low bioavailability of luteolin. Kang Zhiruo (Kang Zhiruo. Preparation and pharmacological evaluation of luteolin nanocomposites [D]. Shenyang Agricultural University) prepared luteolin liposomes by dispersion method: 50 mg of phospholipids, 12.5 mg of cholesterol, 2 mL of luteolin solution with a concentration of 1 mg / mL and anhydrous ethanol were dissolved in a round-bottom flask, anhydrous ethanol was evaporated on a rotary evaporator, phosphate buffer was placed in a round-bottom flask, magnetically stirred after the temperature reached 45°C, and filtered through a 0.45 μm filter membrane to obtain luteolin liposomes; the encapsulation rate of luteolin liposomes obtained by this method was only 89.56%, and the encapsulation rate effect was still low, and the product particle size was large and not easily absorbed.

[0008] Chinese invention patent CN117159493A discloses a chitosan quaternary ammonium salt luteolin liposome and its preparation method and application. The preparation method is: dissolve luteolin in methanol, stir at room temperature until completely dissolved to obtain solution A; dissolve soybean lecithin and cholesterol in chloroform, stir at room temperature until completely dissolved to obtain solution B; pour solution A into solution B, stir at room temperature until there is no particle, add Tween 80 and stir until transparent, after the solvent is completely evaporated, add ultrapure water to dissolve; ultrasonic vibration to obtain luteolin nanoliposome aqueous solution; mix the luteolin nanoliposome aqueous solution with the chitosan quaternary ammonium salt solution evenly to obtain chitosan quaternary ammonium salt luteolin liposome with anti-Helicobacter pylori. However, the invention uses a variety of organic solvents such as methanol and chloroform in the preparation process of luteolin liposomes, and the obtained product has certain hazards.

[0009] In view of this, the present invention provides a method for preparing luteolin liposomes which is green, simple, free of harmful reagents, has high encapsulation rate, small particle size, uniform particle size, high stability, and significant sustained-release effect. The obtained luteolin liposomes can be used in the preparation of cosmetics with good sustained-release effect. Summary of the invention

[0010] In view of the problems existing in the prior art, the present invention provides a method for preparing luteolin liposomes, a product and an application thereof. The luteolin liposomes are prepared by controlling the mass ratio of lecithin, cholesterol and luteolin, controlling the pH value, hydration time and temperature of the hydration solvent, and adopting a suspension treatment process with specific parameters. The obtained luteolin liposomes have high encapsulation rate and low particle size, high stability and good sustained-release effect.

[0011] To achieve the above purpose, the technical solution adopted by the present invention is as follows:

[0012] First, the present invention provides a method for preparing luteolin liposomes, comprising the steps of:

[0013] S1, dissolving lecithin, cholesterol and luteolin in anhydrous ethanol at a mass ratio of 40-60:15:4-6, and drying under reduced pressure to form a film;

[0014] S2. Hydration: The film is mixed with phosphate buffer solution (PBS) at pH=6.8-7.2 or water, stirred and hydrated at 50-60° C. for 6-24 hours to form a suspension, and the suspension is subjected to probe ultrasound to obtain luteolin liposomes.

[0015] Preferably, in step S1, the mass ratio of lecithin, cholesterol and luteolin is 60:15:4-6.

[0016] Further preferably, in step S1, the mass ratio of lecithin, cholesterol and luteolin is 60:15:4.

[0017] Preferably, in step S1, the ratio of the total mass of the lecithin, cholesterol and luteolin to the volume of anhydrous ethanol is 59-81:20 mg / mL.

[0018] Further preferably, in step S1, the ratio of the total mass of the lecithin, cholesterol and luteolin to the volume of anhydrous ethanol is 79-81:20 mg / mL.

[0019] More preferably, in step S1, the ratio of the total mass of the lecithin, cholesterol and luteolin to the volume of anhydrous ethanol is 79:20 mg / mL.

[0020] Preferably, in step S1, the reduced pressure spin drying is specifically: reduced pressure spin drying at 40-55°C.

[0021] Further preferably, in step S1, the reduced pressure spin drying is specifically: reduced pressure spin drying at 45°C.

[0022] Preferably, in step S2, the pH of the phosphate buffer is 7.2.

[0023] Preferably, in step S2, the solid-liquid ratio of the suspension is 59-81 mg / mL.

[0024] Further preferably, in step S2, the solid-liquid ratio of the suspension is 79-81:100 mg / mL

[0025] More preferably, in step S2, the solid-liquid ratio of the suspension is 79:100 mg / mL.

[0026] Preferably, in step S2, the composition of the phosphate buffer is: NaCl, KCl, Na 2 HPO 4 and KH 2 PO4 ; NaCl, KCl, Na 2 HPO 4 and KH 2 PO 4 The mass ratio is 8:0.2:1.44:0.24.

[0027] Preferably, in step S2, the molar concentration of the phosphate buffer is 0.005-0.02M.

[0028] Further preferably, in step S2, the molar concentration of the phosphate buffer is 0.01M.

[0029] Preferably, in step S2, the stirring speed of the hydration is 600-2000 r / min; further preferably, the stirring speed of the hydration is 1100 r / min.

[0030] Preferably, in step S2, the hydration is specifically: stirring and hydrating at 60° C. for 12 hours.

[0031] Preferably, in step S2, the probe ultrasound is specifically: power 200-300kW, probe ultrasound 3-10min.

[0032] Further preferably, in step S2, the probe ultrasound is specifically: power 260kW, probe ultrasound 5min.

[0033] Then, the present invention provides a luteolin liposome prepared by the above preparation method.

[0034] Preferably, the luteolin liposomes of the present invention have an encapsulation efficiency of 86.6%-93.95% and an average particle size of 156.94nm-208.90nm.

[0035] Furthermore, the present invention provides a lyophilized luteolin liposome, comprising components: luteolin liposome and a lyophilization protectant; the lyophilization protectant is lactose.

[0036] Preferably, in the freeze-drying of the luteolin liposome, the mass ratio of the luteolin liposome to the lyoprotectant is 3-8:1.5.

[0037] Further preferably, the mass ratio of the luteolin liposome to the lyoprotectant is 5:1.5.

[0038] Preferably, the preparation method of the lyophilized luteolin liposome is: mixing the luteolin liposome with a lyophilization protectant, pre-freezing and then freeze-drying to obtain the lyophilized luteolin liposome.

[0039] Further preferably, the pre-freezing condition is: pre-freezing at -80 to -60°C for 20 to 50 minutes.

[0040] More preferably, the pre-freezing condition is: pre-freezing at -80°C for 30 minutes.

[0041] More preferably, the freeze-drying time is 20-36 h; even more preferably, the freeze-drying time is 24 h.

[0042] Finally, the present invention provides the use of the luteolin liposomes prepared by the above preparation method in cosmetics.

[0043] Preferably, the cosmetic is a cosmetic that soothes the skin and has no irritating effect on sensitive skin.

[0044] Compared with the prior art, the present invention has the following beneficial effects:

[0045] 1. The present invention prepares luteolin liposomes by controlling the mass ratio of lecithin, cholesterol and luteolin, controlling the pH of the hydration solvent, the hydration time and the temperature, and controlling the process parameters such as the suspension treatment. The obtained luteolin liposomes have high encapsulation rate and low particle size, high stability and good sustained-release effect.

[0046] 2. The present invention encapsulates luteolin in liposomes, so that the encapsulated luteolin is more stable and not easy to precipitate; when the luteolin liposomes are applied to cosmetics, luteolin can be better absorbed through the skin and exert a more lasting effect in the deep layer of the skin with the help of the characteristics of the liposomes; the luteolin liposomes prepared by the present invention not only improve the stability of the cosmetic products, but also improve the bioavailability of luteolin. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] Figure 1 It is a schematic diagram of the structure of luteolin liposomes of the present invention.

[0048] Figure 2 This is the particle size distribution diagram of the luteolin liposomes prepared in Example 1. DETAILED DESCRIPTION

[0049] The following non-limiting examples can enable those skilled in the art to more fully understand the present invention, but do not limit the present invention in any way. The following content is merely an exemplary description of the scope of the present invention, and those skilled in the art can make various changes and modifications to the present invention based on the disclosed content, which should also fall within the scope of the present invention.

[0050] When the embodiments give numerical ranges, it should be understood that, unless otherwise specified in the present invention, both endpoints of each numerical range and any numerical value between the two endpoints can be selected. Unless otherwise defined, all technical and scientific terms used in the present invention have the same meanings as those generally understood by those of ordinary skill in the art to which the present invention belongs.

[0051] The present invention is further described below by way of specific examples. Unless otherwise specified, the various chemical reagents used in the examples of the present invention are obtained through conventional commercial channels.

[0052] In the following examples, the lecithin was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd., with the item number L105734; the cholesterol was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd., with the item number C104029. For the above components, products from different manufacturers have no significant effect on the effects.

[0053] The schematic diagram of the structure of luteolin liposome prepared by the present invention is shown in Figure 1 .

[0054] Example 1

[0055] A method for preparing luteolin liposomes is as follows:

[0056] S1. Dissolve lecithin, cholesterol and luteolin in 20 mL of anhydrous ethanol at a mass ratio of 60 mg:15 mg:4 mg; after dissolution, spin dry under reduced pressure at 45° C. in a round-bottom flask to form a thin film.

[0057] S2. Hydration: The film of step S1 was mixed with 100 mL of pH = 7.2 0.01 M phosphate buffer (PBS), and stirred and hydrated at 60°C and 1100 r / min for 12 h to form a uniformly dispersed suspension. The suspension was subjected to probe ultrasonication for 5 min at a probe ultrasonic power of 260 kW to obtain luteolin liposomes (i.e., luteolin liposome suspension).

[0058] The composition of the phosphate buffer is: NaCl, KCl, Na 2 HPO 4 and KH 2 PO 4 ; NaCl, KCl, Na 2 HPO 4 and KH 2 PO 4 The mass ratio is 8:0.2:1.44:0.24.

[0059] The particle size distribution of the luteolin liposomes prepared in this example is shown in Figure 2 .

[0060] Example 2

[0061] The difference from Example 1 is that in step S1, the mass ratio of lecithin, cholesterol and luteolin is 60:15:6. The rest is the same as Example 1.

[0062] Example 3

[0063] The difference from Example 1 is that in step S1, the mass ratio of lecithin, cholesterol and luteolin is 40:15:4. The rest is the same as Example 1.

[0064] Example 4

[0065] The difference from Example 1 is that in step S2, the hydration time is 6 hours. The rest is the same as Example 1.

[0066] Example 5

[0067] The difference from Example 1 is that the phosphate buffered saline (PBS) in step S2 is replaced with deionized water without pH adjustment. The rest is the same as Example 1.

[0068] Comparative Example 1

[0069] The difference from Example 1 is that in step S1, the mass ratio of lecithin, cholesterol and luteolin is 120:15:4. The rest is the same as Example 1.

[0070] Comparative Example 2

[0071] The difference from Example 1 is that in step S1, the mass ratio of lecithin, cholesterol and luteolin is 60:15:10. The rest is the same as Example 1.

[0072] Comparative Example 3

[0073] The difference from Example 1 is that in step S2, the pH of the phosphate buffer used is 7.4. The rest is the same as Example 1.

[0074] Comparative Example 4

[0075] The difference from Example 1 is that in step S2, the hydration time is 4 hours. The rest is the same as Example 1.

[0076] Comparative Example 5

[0077] The difference from Example 1 is that in step S2, the hydration temperature is 70° C. The rest is the same as Example 1.

[0078] Comparative Example 6

[0079] The difference from Example 1 is that in step S2, the hydration temperature is 30° C. The rest is the same as Example 1.

[0080] Comparative Example 7

[0081] The difference from Example 1 is that the probe ultrasonic treatment is not performed, specifically:

[0082] S1. Dissolve lecithin, cholesterol and luteolin in 20 mL of anhydrous ethanol at a mass ratio of 60 mg:15 mg:4 mg; after dissolution, spin dry under reduced pressure at 45° C. in a round-bottom flask to form a thin film.

[0083] S2. Hydration: The film of step S1 was mixed with 100 mL of pH=7.2 0.01 M phosphate buffer (PBS), and stirred and hydrated at 60° C. and 1100 r / min for 12 h to form a uniformly dispersed suspension, thereby obtaining luteolin liposomes (luteolin liposome suspension).

[0084] Comparative Example 8

[0085] The difference from Example 1 is that in step S1, the mass ratio of lecithin, cholesterol and luteolin is 100:25:4. The rest is the same as Example 1.

[0086] Test 1: Particle size and encapsulation rate detection

[0087] Encapsulation efficiency detection method: The luteolin liposome sample was centrifuged at 2500r / min for 6min to separate the free drug. Then, 0.2mL of the supernatant was vortexed with 2mL of methanol for 10min to break the emulsion so that all the encapsulated luteolin was released. The ultraviolet absorbance at 347nm was detected to calculate the encapsulation efficiency of the luteolin liposome.

[0088] Encapsulation efficiency (%) = (W en -W total )×100%; where W en W is the amount of drug encapsulated in the luteolin liposome suspension after centrifugation and demulsification (mg); total It is the amount of drug encapsulated in the luteolin liposome suspension measured before demulsification by centrifugation (mg).

[0089] Particle size detection method: Take a luteolin liposome sample and dilute it to 200 times with physiological saline; take 2 mL of the diluted sample and measure the liposome particle size distribution using a laser particle size analyzer by dynamic light scattering method.

[0090] The average particle size and encapsulation efficiency test results of the luteolin liposomes prepared in each example and comparative example are shown in Table 1.

[0091] Table 1

[0092] Average particle size (nm) Encapsulation rate (%) Example 1 179.93 93.95 Example 2 184.73 91.19 Example 3 156.94 89.12 Example 4 208.90 86.36 Example 5 185.19 92.48 Comparative Example 1 260.92 66.60 Comparative Example 2 288.32 77.54 Comparative Example 3 142.17 69.01 Comparative Example 4 187.21 59.37 Comparative Example 5 189.59 80.64 Comparative Example 6 225.67 60.83 Comparative Example 7 1305.99 89.08 Comparative Example 8 246.33 89.40

[0093] It can be seen from Table 1 that the luteolin liposomes prepared by the preparation method of the embodiments of the present invention have a high encapsulation rate, which is 86.60%-93.95%; in addition, the average particle size of the luteolin liposomes in each embodiment is 156.94nm-208.90nm, and the particle size is small and uniform, which is conducive to efficient absorption in the application of cosmetics.

[0094] Experiment 2: In vitro release experiment

[0095] Experimental method: With reference to the Chinese Pharmacopoeia 2020 edition, it was verified that the luteolin liposomes of the present invention have a sustained-release effect.

[0096] 2 mL of the luteolin liposome suspension of Example 1 was placed in 30 mL of PBS solution (0.01 M, pH 7.4), released at 37°C and 100 rpm / min, and 2 mL of the supernatant was taken at 0.5 h, 1 h, 4 h, 8 h, 12 h, 24 h, 48 h, 72 h, 96 h, and 120 h, and 2 mL of PBS was added after taking it out. The obtained supernatant was subjected to UV-visible spectrophotometric detection at a detection wavelength of 347 nm.

[0097] Control group: 2 mL of PBS solution with only luteolin dissolved was placed in 30 mL of PBS solution (0.01 M, pH 7.4), and released at 37°C and 100 rpm / min. 2 mL of supernatant was taken at 0.5 h, 1 h, 4 h, 8 h, 12 h, and 24 h, and 2 mL of PBS was added after taking it out. The supernatant was detected by UV-visible spectrophotometry at a wavelength of 347 nm.

[0098] The results of in vitro cumulative release rate are shown in Table 2.

[0099] Table 2

[0100]

[0101] It can be seen from Table 2 that the luteolin liposomes obtained by the preparation method of the embodiment of the present invention have a good sustained-release effect.

[0102] Test 3: Stability test

[0103] Experimental method: Take 10 mL of liposome suspension and place it in 50℃, 25℃ and 4℃ environments respectively. Take samples on the 1st, 3rd, 5th, 7th, 15th and 30th days to measure the encapsulation efficiency and particle size changes of the samples. The results are shown in Table 3.

[0104] Table 3

[0105]

[0106] As can be seen from Table 3, after the luteolin liposomes prepared by the present invention were stored at 4°C for 30 days, the encapsulation efficiency was better than that at 50°C and 25°C; it can be seen that at 4°C, the luteolin liposomes have good stability in terms of the effect of the encapsulation efficiency. The luteolin liposomes prepared by the present invention were stored at 50°C, 25°C, and 4°C, and the particle size characterization trends were similar.

[0107] Experiment 4: Lyophilization and reconstitution experiment

[0108] Experimental method: Take 5mL of luteolin liposomes in a sample bottle, add 1.5mL of lactose freeze-drying protective agent and mix thoroughly, place in -80℃ refrigerator for pre-freezing for 30min, transfer to freeze dryer, freeze-dry for 24h until powder, add 5mL of 0.01M PBS (pH 7.0) to the freeze-dried sample, ultrasonicate until completely dissolved, and then measure the change in particle size. The particle size results after re-dissolving for 1 day and 3 days are shown in Table 4.

[0109] Table 4

[0110]

[0111] It can be seen from Table 4 that the luteolin liposomes prepared by the present invention have a better re-dissolution effect when lactose is used as a lyoprotectant. The particle size of the luteolin liposomes after freeze-drying and re-dissolution is still smaller than that of the group without lyoprotectant, and the particle size has not changed significantly.

[0112] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, rather than to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions of the technical solution of the present invention by ordinary technicians in this field do not deviate from the essence and scope of the technical solution of the present invention.

Claims

1. A method for preparing luteolin liposomes, characterized in that: Includes steps: S1, dissolving lecithin, cholesterol and luteolin in anhydrous ethanol at a mass ratio of 40-60:15:4-6, and drying under reduced pressure to form a film; S2. Hydration: Mix the film with a phosphate buffer solution or water at pH=6.8-7.2, stir and hydrate at 50-60° C. for 6-24 hours to form a suspension, and perform probe ultrasound on the suspension to obtain luteolin liposomes.

2. The preparation method according to claim 1, characterized in that: In step S1, the mass ratio of lecithin, cholesterol and luteolin is 60:15:4-6.

3. The preparation method according to claim 2, characterized in that: In step S1, the mass ratio of lecithin, cholesterol and luteolin is 60:15:

4.

4. The preparation method according to claim 1, characterized in that: In step S1, the ratio of the total mass of lecithin, cholesterol and luteolin to the volume of anhydrous ethanol is 59-81:20 mg / mL; in step S1, the reduced pressure spin drying is specifically: reduced pressure spin drying at 40-55°C.

5. The preparation method according to claim 1, characterized in that: In step S2, the pH of the phosphate buffer is 7.2; in step S2, the solid-liquid ratio of the suspension is 59-81 mg / mL; and the molar concentration of the phosphate buffer is 0.005-0.02M.

6. The preparation method according to claim 1, characterized in that: In step S2, the stirring speed of the hydration is 600-2000 r / min; the hydration is specifically: stirring and hydrating at 60° C. for 12 hours.

7. The preparation method according to claim 1, characterized in that: In step S2, the probe ultrasound is specifically: power 200-300kW, probe ultrasound 3-10min.

8. A luteolin liposome, characterized in that: The luteolin liposomes obtained by the preparation method according to any one of claims 1 to 7 have an encapsulation rate of 86.6%-93.95% and an average particle size of 156.94nm-208.90nm.

9. A lyophilized luteolin liposome, characterized in that: The invention comprises the following components: the luteolin liposomes according to claim 8 and a freeze-drying protective agent; the freeze-drying protective agent is lactose; and the mass ratio of the luteolin liposomes to the freeze-drying protective agent is 3-8:1.

5.

10. Application of luteolin liposomes in cosmetics, characterized in that: The luteolin liposome is the luteolin liposome obtained by the preparation method according to any one of claims 1 to 7 or the luteolin liposome according to claim 8.

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