A preparation method of quercetin liposome

Abstract

The present invention relates to a method for preparing quercetin liposomes, comprising the following steps: (1) adding quercetin, lecithin, ceramide and cholesterol to a solvent for mixing, and performing vacuum rotary evaporation to obtain an oil phase; (2) preparing a PBS buffer, adding polyethylene glycol, and mixing uniformly to obtain a PBS-PEG solution, i.e., an aqueous phase; (3) adding the aqueous phase to the oil phase, heating in a water bath, and then emulsifying to obtain quercetin liposomes. The quercetin liposomes obtained by the present invention have a small particle size and uniform distribution, a high encapsulation rate, are conducive to transdermal absorption, and have good market application prospects.

Description

Technical Field

[0001] The invention belongs to the field of natural plant preparations, and particularly relates to a method for preparing quercetin liposomes. Background Art

[0002] Quercetin is a plant flavonol widely found in natural plants. It has pharmacological effects such as anti-free radical, anti-oxidation, antibacterial, anti-viral, anti-inflammatory, anti-allergic, anti-ultraviolet damage, anti-aging, sunscreen, whitening, and inhibition of skin scar formation. According to current research, quercetin has low toxicity. The LD50 of oral administration to mice is about 10-50g / kg. Long-term application has no toxic reaction and no obvious skin irritation. In view of the wide potential skin care effects of quercetin, many countries have included quercetin in the catalog of cosmetic raw materials in recent years. The commercially available cosmetics containing quercetin are mainly creams, lotions, essences, toners, etc. Among them, the compatibility of quercetin and kojic acid can enhance the stability of kojic acid.

[0003] However, the pharmaceutics characteristics of quercetin are not ideal, and the main problems include: quercetin has poor in vitro stability (light instability, acid-base instability). Therefore, by using pharmaceutical methods, developing a suitable carrier for quercetin to improve its stability in application has high potential and value. As a new type of carrier, liposomes have the characteristics of improving the stability of the main drug. Although there has been some progress in the research on the preparation of quercetin liposomes (CN202010879360.9, CN201110453651.2, CN201210590103.9), the particle size of the quercetin liposomes currently prepared is still large, with a particle size of about 200nm, which is not conducive to transdermal absorption (CN201810376603.X). Summary of the invention

[0004] The technical problem to be solved by the present invention is to provide a method for preparing quercetin liposomes. The quercetin liposomes obtained by the method have small particle size and uniform distribution, high encapsulation rate, are conducive to transdermal absorption, and have good market application prospects.

[0005] The present invention provides a method for preparing quercetin liposomes, comprising the following steps:

[0006] (1) adding quercetin, lecithin, ceramide and cholesterol into a solvent, mixing, and performing rotary evaporation under reduced pressure to obtain an oil phase;

[0007] (2) preparing PBS buffer, adding polyethylene glycol, and mixing well to obtain a PBS-PEG solution, i.e., an aqueous phase;

[0008] (3) adding the aqueous phase to the oil phase, heating in a water bath, and then emulsifying to obtain quercetin liposomes.

[0009] The mass ratio of quercetin, lecithin and ceramide in the step (1) is 1-2:35-40:1-2, and the molar ratio of cholesterol to ceramide is 1-2:1-2.

[0010] The ceramide-like substance is one or more of Cetyl-PG hydroxyethyl palmitamide and arachidonic acid amide MEA.

[0011] The solvent in step (1) is one or more of chloroform, ethanol, and ether; the mass volume ratio of quercetin to the solvent is 1-2g:200-300mL.

[0012] The PBS buffer concentration in step (2) is 20-30 mmol / L, and the pH value is 7.

[0013] The polyethylene glycol in step (2) is one or more of PEG200 and PEG400; the volume ratio of the polyethylene glycol to the PBS buffer is 1-2:20-30.

[0014] The water bath heating temperature in step (3) is 35-40° C. and the heating time is 1-2 h.

[0015] The emulsification speed in the step (3) is 6000-8000 r / min, and the emulsification time is 30 min.

[0016] After emulsification in step (3), the mixture is stable at 4°C for 2-3 hours.

[0017] Ceramides are a class of chemicals used to treat skin problems. They improve skin condition by repairing skin tissue, increasing the skin's ability to retain moisture, and reducing skin sensitivity.

[0018] Beneficial Effects

[0019] The quercetin liposome obtained by the invention has small particle size and uniform distribution, high encapsulation rate, is conducive to transdermal absorption, and has good market application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 The particle size distribution of the quercetin liposomes obtained in Example 1;

[0021] Figure 2 The in vitro transdermal absorption results of the quercetin liposomes obtained in Example 1 and the control group are shown. DETAILED DESCRIPTION

[0022] The present invention will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and are not intended to limit the scope of the present invention. In addition, it should be understood that after reading the content taught by the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms fall within the scope limited by the appended claims of the application equally.

[0023] Example 1

[0024] (1) Preparation of the oil phase:

[0025] 1. Weigh 2g of quercetin, 36g of lecithin and 2g of Cetyl-PG hydroxyethyl palmitamide: cholesterol (1:1 molar ratio) and place them in a rotary evaporator. Add about 300ml of chloroform and stir to dissolve by ultrasonic until no obvious solids remain.

[0026] 2. Place the rotary evaporator on a rotary evaporator and evaporate under reduced pressure for 5 hours at a water bath temperature of 35°C. Evaporate the chloroform in the solution until no more chloroform comes out. At this time, there is a uniform film layer on the inner wall of the rotary evaporator, which is the oil phase.

[0027] (2) Preparation of the aqueous phase:

[0028] Prepare 200 ml of 20 mmol / L PBS buffer (pH 7.0), add 10 ml of PEG400 to the above solution, mix well to obtain a PBS-PEG solution, i.e., the aqueous phase.

[0029] (3) Emulsification:

[0030] 1. Add the above 200 ml of water phase to the oil phase, shake to mix, and heat in a water bath with rotation at 35°C for 1 hour. If some solids are still not dissolved, then ultrasonicate at room temperature until there is no obvious solid, and store at 4°C overnight.

[0031] 2. Use a laboratory emulsifier at a speed of 7000 r / min to fully emulsify for about 30 minutes. Finally, stabilize at 4°C for 2 hours to obtain quercetin liposomes.

[0032] (4) Characterization:

[0033] The prepared quercetin liposome sample was filtered using a 1 μm PES filter, which was difficult to filter. About 2 / 3 of the sample was centrifuged at 5000 r / min for 10 min, and the sample appeared in three layers (the upper layer was a transparent liquid layer, the middle layer was a white emulsified layer, and the lower layer was a yellow solid layer, which should be the added quercetin); the sample was ultrasonically mixed, and then emulsified again for about 30 minutes, and then sampled again to measure the particle size distribution, most of which were 24 nm.

[0034] The uncentrifuged samples were directly sampled for characterization analysis, such as Figure 1As shown, the measured particle size distributions of 2789 nm, 264.9 nm, and 44.7 nm accounted for 61.7%, 27.9%, and 10.4%, respectively.

[0035] Test Example 1 Quercetin liposome encapsulation efficiency test

[0036] The free quercetin concentration in the quercetin liposomes obtained in Example 1 and the total quercetin concentration in the liposomes were detected by high performance liquid chromatography, and then the encapsulation efficiency of the quercetin liposomes was calculated.

[0037] The experimental conditions are as follows

[0038] Chromatographic column: phenyl column, 5μm, 100A;

[0039] Flow rate: 1.0 ml / min; injection volume: 10 μl; column temperature: 30 °C;

[0040] Mobile phase A: 0.2% formic acid water, mobile phase B: methanol; detection wavelength: 360nm;

[0041] Gradient elution conditions: 45% A 55% B isocratic elution for 30 min.

[0042] Blank solvent: methanol;

[0043] Control solution: weigh 10 mg of sample, add 10 ml of methanol, shake to dissolve, and filter (0.22 um filter head).

[0044] Sample solution 1 (quercetin supernatant): The supernatant of quercetin liposomes after standing for 2-3 days was taken and diluted 10 times with methanol, then sonicated and filtered (0.22 um filter head).

[0045] Sample solution 2 (quercetin liposomes): the liposomes were ultrasonically mixed again after standing, diluted 20 times with methanol, ultrasonically filtered (0.22 um filter head).

[0046] The free concentration of quercetin was measured to be 0.13 mg / ml under the above conditions; the overall concentration of quercetin liposomes was 6.49 mg / ml.

[0047] The formula for calculating the liposome encapsulation efficiency is EN% = (1-Cf / Ct) × 100%; Cf is the free drug concentration; Ct is the total amount of drug in the liposome suspension.

[0048] The encapsulation efficiency of the quercetin liposome obtained in Example 1 was 98%.

[0049] Test Example II Quercetin liposome in vitro transdermal test

[0050] The experimental group was the quercetin liposomes prepared in Example 1, and the composition table is as follows:

[0051] No. Material Name Proportion (%) 1 Quercetin 0.86 2 Hydrogenated Lysolecithin 15.57 3 Cetyl-PG Hydroxyethyl Palmitamide (Ceramide E) 0.86 4 cholesterol 0.56

[0052] The control group was quercetin liposomes prepared according to the literature (Liu D, Hu H, Lin Z, et al. Quercetin deformable liposome: preparation and efficacy against ultraviolet B induced skin damages in vitro and in vivo [J]. Journal of Photochemistry and Photobiology B: Biology, 2013, 127: 8-17), and the composition ratio is as follows:

[0053] No. Material Name Proportion (%) 1 Quercetin 0.86 2 Phosphatidylcholine 16 3 Twain 80 4 4 cholesterol 4

[0054] Using the RYJ-12B drug transdermal diffusion tester, cut a piece of intact skin of appropriate size and fix it in the diffusion pool, so that the stratum corneum faces the supply pool and the dermis faces the diffusion pool. Inject 15ml of physiological saline into the receiving chamber, add 1ml of the above liposomes to the supply pool respectively, and use an iron clamp to fix the joint of the supply pool and the receiving pool in a constant temperature water bath of (37.0±0.1)℃, and keep the magnetic stirring at about 500r·min -1 , remove all the solution in the receiving chamber at 1h, 3, 5, 7, 9, 12, 24h, and 30h of equilibrium, and immediately add fresh receiving solution of the same volume and temperature (pay attention to keep the subcutaneous layer in close contact with the liquid surface and remove all bubbles). Filter the sample solution with a 0.22μm microporous filter membrane, discard the first 1mL of the primary filtration solution, accurately draw 20μL of the subsequent filtrate, and determine the quercetin content in the diffusion solution according to the conditions of high performance liquid chromatography [Yi Yin, Liu Ning. High performance liquid chromatography method for determining the content of quercetin in plants [J]. Plant Physiology Communications, 2000, 36(5): 445-446].

[0055] The cumulative permeation per unit area at each time point (Q, μg·cm) was calculated according to the following formula: 2 )

[0056]

[0057] Qn is the drug mass concentration (μg / ml) measured at the nth sampling point; Ci is the drug mass concentration (μg / ml) measured at the ith (i≤n-1) sampling point; A: effective transdermal area (1.33cm 2 ); V is the volume of receiving liquid in the receiving tank (ml).

[0058] The results are as follows Figure 2 As shown by Figure 2 It can be seen that the in vitro permeability of the quercetin liposomes in Example 1 is significantly higher than that of the quercetin liposomes in the control group.

Claims

1. A method for preparing quercetin liposomes, comprising the following steps: (1) adding quercetin, lecithin, ceramide and cholesterol to a solvent, mixing, and performing vacuum rotary evaporation to obtain an oil phase; wherein: The mass ratio of quercetin, lecithin and ceramide is 1-2:35-40:1-2, the molar ratio of cholesterol to ceramide is 1-2:1-2; the ceramide is Cetyl-PG hydroxyethyl palmitamide; (2) Prepare PBS buffer, add polyethylene glycol, and mix well to obtain PBS-PEG solution, i.e., aqueous phase; (3) The aqueous phase is added to the oil phase, heated in a water bath, and then emulsified to obtain quercetin liposomes.

2. The preparation method according to claim 1, characterized in that: The solvent in step (1) is one or more of chloroform, ethanol, and ether; the mass volume ratio of quercetin to the solvent is 1-2 g: 200-300 mL.

3. The preparation method according to claim 1, characterized in that: The PBS buffer concentration in step (2) is 20-30 mmol / L, and the pH value is 7.

4. The preparation method according to claim 1, characterized in that: The polyethylene glycol in step (2) is PEG400; the volume ratio of the polyethylene glycol to the PBS buffer is 1-2:20-30.

5. The preparation method according to claim 1, characterized in that: The water bath heating temperature in step (3) is 35-40° C. and the heating time is 1-2 h.

6. The preparation method according to claim 1, characterized in that: The emulsification speed in step (3) is 6000-8000 r / min, and the emulsification time is 30 min.

7. The preparation method according to claim 1, characterized in that: After emulsification in step (3), the mixture is stable at 4°C for 2-3 hours.

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