Preparation process of peony root bark extract liposome

Abstract

The invention discloses a preparation process of peony root bark extract liposome, which comprises the following steps: S1, dissolving 0.1 g of peony root bark extract in water to obtain a phase A of a mixed solution; s2, adding 2g of PC-80, 0.4 g of cholesterol and 0.2 g of VE into ethanol, and stirring to obtain a mixed solution phase B; s3, slowly adding the phase A in the homogenizing process of the phase B; s4, carrying out rotary evaporation to remove ethanol, and then adding pentanediol and glycerol; s5, obtaining peony root bark extract lipidosome, and storing the peony root bark extract lipidosome in a dark place at low temperature; the liposome is prepared by adopting a multiple emulsion method, and the preparation process of the liposome is optimized by taking the encapsulation efficiency and the stability as indexes. A liposome preparation process is simplified, and introduction of an organic solvent is avoided.

Description

Technical Field

[0001] The invention relates to the technical field of preparation of peony root bark extract, in particular to a preparation process of peony root bark extract liposome. Background Art

[0002] Peony (Paeonia suffruticosa Andr.) is a shrub of the genus Paeonia in the family Ranunculaceae. It is a traditional precious flower with a planting area of ​​about 200 km in my country. 2 , mainly distributed in the Yangtze River and Yellow River basins such as Shanxi, Henan, Shandong, Anhui and other places. The application of peony-related products in cosmetics, in addition to a variety of forms, also involves a wide range of functions, including whitening, anti-aging, anti-inflammatory, antioxidant, etc., with broad prospects. Literature research shows that extracts from all parts of peony have shown a positive effect on the pathways related to melanin production and accumulation, and the development prospects for their application in whitening products are very broad.

[0003] However, peony root bark extract has some limitations in formula application. 1) Poor stability: Some active ingredients have poor stability and must be stored under specific conditions to maintain their activity. 2) Uncertainty of transdermal absorption: Due to the complex composition of peony root bark extract, the amount of transdermal absorption is difficult to measure, which further increases the uncertainty of efficacy. 3) Appearance limitations: Due to the color of the extract itself, it is difficult to increase its dosage, so its application in the formula remains conceptual and it is difficult to achieve the claimed efficacy.

[0004] Liposomes, also known as lipid globules or liquid crystal microcapsules, are microscopic spherical carriers formed by encapsulating active ingredients in lipid bilayers. The water-soluble components can be encapsulated in the inner water phase of phospholipids, and the fat-soluble components can be encapsulated between the phospholipid bilayers. The basic components of liposomes are usually amphiphilic phospholipids and cholesterol. Amphiphilic phospholipids form a bilayer structure, and cholesterol supports and maintains the bilayer structure. Liposomes have a similar structure to cell membranes, which can promote the penetration of active ingredients into the skin, enhance the degree of entry of active ingredients into the stratum corneum or intercellular lipids, increase the accumulation of active ingredients in the local skin, thereby improving bioavailability, reducing adverse reactions, and having a sustained release effect. Therefore, the problem of difficult absorption of active ingredients can be solved through liposome encapsulation technology, and water-soluble and fat-soluble ingredients can be carried to the deep layers of the skin to exert their active effects. Summary of the invention

[0005] The purpose of the present invention is to provide a preparation process of peony root bark extract liposomes to solve the problems raised in the above background technology.

[0006] In order to achieve the above object, the present invention adopts the following technical scheme: a preparation process of liposomes of peony root bark extract, comprising the following steps:

[0007] S1, dissolving 0.1 g of peony root bark extract in water to obtain a mixed solution phase A;

[0008] S2, adding 2 g PC-80, 0.4 g cholesterol and 0.2 g VE to ethanol, stirring to obtain a mixed solution phase B;

[0009] S3. During the homogenization of phase B, slowly add phase A;

[0010] S4, remove ethanol by rotary evaporation, and then add pentanediol and glycerol;

[0011] S5. Obtain peony root bark extract liposomes and store them at low temperature and away from light.

[0012] Preferably, the temperature of water in step S1 is 70°C.

[0013] Preferably, the stirring temperature in step S2 is 60°C.

[0014] Preferably, the homogenization conditions of phase B in step S3 are 2000 rpm and 5 min.

[0015] The present invention provides a method for detecting the encapsulation efficiency of liposomes of peony root bark extract, comprising the following steps:

[0016] S11, accurately pipette 0.2 mL of liposome solution;

[0017] S12, add an equal amount of 10 mg / mL protamine and mix thoroughly;

[0018] S13, centrifuge at 4000 rpm / min for 20 min, and take the supernatant to determine the content of peony root bark extract;

[0019] S14. Finally, the encapsulation efficiency is calculated.

[0020] Preferably, in step S14 of the method for detecting the encapsulation efficiency of peony root bark extract liposomes, the encapsulation efficiency is calculated as follows: encapsulation efficiency % = 1-(supernatant peony root bark extract content / total content).

[0021] The present invention provides a method for detecting the stability of peony root bark extract liposomes, comprising the following steps:

[0022] S21, accurately pipette the liposome solution;

[0023] S22, centrifuge at 4000 rpm / min for 20 min, and take the supernatant;

[0024] S23, measuring the OD value of the liposomes before and after centrifugation, and calculating the difference;

[0025] S24. Observe the appearance of liposomes under different conditions.

[0026] Preferably, in step S24 of the stability detection method of peony root bark extract liposomes, the observation conditions are 4°C, room temperature, and 40°C.

[0027] The present invention provides a method for detecting the concentration of liposomes extracted from peony root bark, comprising the following steps:

[0028] S31, using an ELISA reader to perform a full scan to determine the detection wavelength;

[0029] S32. Detect the OD values ​​of samples of different concentrations under the optimal wavelength conditions and establish a standard curve.

[0030] Compared with the prior art, the present invention has the following beneficial effects:

[0031] The present invention adopts the double emulsion method to optimize the preparation process of liposomes by taking encapsulation rate and stability as indicators, simplifies the preparation process of liposomes, and avoids the introduction of organic solvents. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 This is a schematic diagram of the liposome morphology structure of the present invention;

[0033] Figure 2 Schematic diagram of experimental results of the concentration detection method of liposomes of peony root bark extract of the present invention;

[0034] Figure 3 Schematic diagram of the particle size distribution of liposomes of the present invention;

[0035] Figure 4 This is a schematic diagram of melanin comparison of the present invention;

[0036] Figure 5 This is a schematic diagram of the zebrafish whitening model of the present invention. DETAILED DESCRIPTION

[0037] In order to further explain the technical solution of the present invention, it is described in detail below through specific embodiments.

[0038] Example 1

[0039] 1. Dissolve 0.1 parts of peony root bark extract in 41.9 parts of water and stir at 70°C to obtain a mixed solution phase A;

[0040] 2. Add 2 parts of PC-80, 0.4 parts of cholesterol and 0.2 parts of VE to 47.4 parts of ethanol and stir at 60°C to obtain a mixed solution phase B;

[0041] 3. During the homogenization of phase B at 2000 rpm, slowly add phase A and continue homogenizing for 5 minutes;

[0042] 4. After homogenization, remove ethanol by rotary evaporation and add pentanediol and glycerol.

[0043] 5. Obtain peony root bark extract liposomes and store them at low temperature and away from light.

[0044] Example 2

[0045] 1. Dissolve 0.2 parts of peony root bark extract in 41.8 parts of water and stir at 70°C to obtain a mixed solution phase A;

[0046] 2. Add 2 parts of PC-80, 0.4 parts of cholesterol and 0.2 parts of VE to 47.4 parts of ethanol and stir at 60°C to obtain a mixed solution phase B;

[0047] 3. During the homogenization of phase B at 2000 rpm, slowly add phase A and continue homogenizing for 5 minutes;

[0048] 4. After homogenization, remove ethanol by rotary evaporation and add pentanediol and glycerol.

[0049] 5. Obtain peony root bark extract liposomes and store them at low temperature and away from light.

[0050] Example 3

[0051] 1. Dissolve 0.075 parts of peony root bark extract in 41.925 parts of water and stir at 70°C to obtain a mixed solution phase A;

[0052] 2. Add 1.5 parts of PC-80, 0.3 parts of cholesterol and 0.2 parts of VE to 48 parts of ethanol and stir at 60°C to obtain a mixed solution phase B;

[0053] 3. During the homogenization of phase B at 2000 rpm, slowly add phase A and continue homogenizing for 5 minutes;

[0054] 4. After homogenization, remove ethanol by rotary evaporation and add pentanediol and glycerol.

[0055] 5. Obtain peony root bark extract liposomes and store them at low temperature and away from light.

[0056] Example 4

[0057] 1. Dissolve 0.1 parts of peony root bark extract in 31.9 parts of water and stir at 70°C to obtain a mixed solution phase A;

[0058] 2. Add 2 parts of PC-80, 0.4 parts of cholesterol and 0.2 parts of VE to 57 parts of ethanol and stir at 60°C to obtain a mixed solution phase B;

[0059] 3. During the homogenization of phase B at 2000 rpm, slowly add phase A and continue homogenizing for 5 minutes;

[0060] 4. After homogenization, remove ethanol by rotary evaporation and add pentanediol and glycerol.

[0061] 5. Obtain peony root bark extract liposomes and store them at low temperature and away from light.

[0062] Comparative Example 1

[0063] 1. Dissolve 0.067 parts of peony root bark extract in 41.933 parts of water and stir at 70°C to obtain a mixed solution phase A;

[0064] 2. Add 2 parts of PC-80, 0.4 parts of cholesterol and 0.2 parts of VE to 47.4 parts of ethanol and stir at 60°C to obtain a mixed solution phase B;

[0065] 3. During the homogenization of phase B at 2000 rpm, slowly add phase A and continue homogenizing for 5 minutes;

[0066] 4. After homogenization, remove ethanol by rotary evaporation and add pentanediol and glycerol.

[0067] 5. Obtain peony root bark extract liposomes and store them at low temperature and away from light.

[0068] Comparative Example 2

[0069] 1. Dissolve 0.1 parts of peony root bark extract in 61.9 parts of water and stir at 70°C to obtain a mixed solution phase A;

[0070] 2. Add 2 parts of PC-80, 0.4 parts of cholesterol and 0.2 parts of VE to 27.4 parts of ethanol and stir at 60°C to obtain a mixed solution phase B;

[0071] 3. During the homogenization of phase B at 2000 rpm, slowly add phase A and continue homogenizing for 5 minutes;

[0072] 4. After homogenization, remove ethanol by rotary evaporation and add pentanediol and glycerol.

[0073] 5. Obtain peony root bark extract liposomes and store them at low temperature and away from light.

[0074] Comparative Example 3

[0075] 1. Dissolve 0.1 parts of peony root bark extract in 51.9 parts of water and stir at 70°C to obtain a mixed solution phase A;

[0076] 2. Add 2 parts of PC-80, 0.4 parts of cholesterol and 0.2 parts of VE to 37.4 parts of ethanol and stir at 60°C to obtain a mixed solution phase B;

[0077] 3. During the homogenization of phase B at 2000 rpm, slowly add phase A and continue homogenizing for 5 minutes;

[0078] 4. After homogenization, remove ethanol by rotary evaporation and add pentanediol and glycerol.

[0079] 5. Obtain peony root bark extract liposomes and store them at low temperature and away from light.

[0080] The present invention provides a method for detecting the concentration of liposomes extracted from peony root bark, comprising the following steps:

[0081] S31, use the microplate reader to perform a full scan to determine that the detection wavelength is 310nm ( Figure 2 center left);

[0082] S32, detect the OD values ​​of samples of different concentrations under the optimal wavelength conditions, and establish a standard curve ( Figure 2 (middle right)

[0083] The specific experimental results are as follows Figure 2 shown.

[0084] The present invention provides a method for detecting the encapsulation efficiency of liposomes of peony root bark extract, comprising the following steps:

[0085] Accurately pipette 0.2 mL of liposome solution; then add an equal amount of 10 mg / mL protamine and mix thoroughly; centrifuge at 4000 rpm / min for 20 min, take the supernatant to determine the content of peony root bark extract; finally calculate the encapsulation efficiency.

[0086] Encapsulation efficiency % = 1-(supernatant peony root bark extract content / total content).

[0087] The present invention provides a method for detecting the stability of peony root bark extract liposomes, comprising the following steps:

[0088] Accurately pipette the liposome solution; centrifuge at 4000 rpm / min for 20 min, take the supernatant to measure the OD value of the liposome before and after centrifugation, and calculate the difference;

[0089] The results of the encapsulation efficiency and stability experiments are as follows:

[0090] Encapsulation rate% Stability difference Example 1 79.73 0.046 Example 2 75.68 0.052 Example 3 74.80 0.052 Example 4 74.20 0.069 Comparative Example 1 56.48 0.160 Comparative Example 2 52.11 0.117 Comparative Example 3 47.62 0.119

[0091] Take 0.2mL of the sample of Example 1 and dilute it with 1mL of deionized water, then put it into the sample cell and place it in the MalvernZetasizer Nano ZS90 series laser particle size analyzer to measure the particle size and Zeta potential of the liposomes. The measurement temperature is 25°C, and the number of measurement cycles is automatically set by the system (n=3). The experimental results show that the average particle size of the sample of Example 1 is 174.5nm and the Zeta potential is -36.7mV. The results show that the liposome particle size distribution is uniform and has good electronegativity.

[0092] 10 mL of the sample of Example 1 was added dropwise to the copper mesh for 1 min, and the floating liquid was removed by filter paper. 10 mL of uranyl acetate was added dropwise to the copper mesh for 1 min, and the floating liquid was removed by filter paper. Dry at room temperature for several minutes. Electron microscopy was performed at 80 kV, and the sample was observed under a transmission electron microscope (Hitachi HT7800), and the image was collected for analysis. The results are shown in the figure. Figure 1 As shown, it can be seen that the liposome has a complete morphological structure and is spherical.

[0093] The formation, transfer, deposition and metabolism of melanin are key factors in determining skin color. The skin structure and melanin synthesis mechanism of zebrafish are highly similar to those of humans. In addition, zebrafish embryos are transparent, with obvious melanin on the body surface, which is easy to observe and present; the embryos develop rapidly, and melanin is rapidly formed within 3 to 4 days, so the test cycle is short. This test intends to use the method of transdermal absorption for sampling, and by comparing the melanin content on the surface of zebrafish, determine the whitening effect of the raw materials.

[0094] Ten 4hpf wild-type AB strain zebrafish fertilized eggs were randomly selected from each well of a 24-well plate, and every three wells formed an experimental group. The samples were diluted with fish water and added at a dilution of 1:1000. The volume of each well was 1mL. After treatment at 28°C for 48 hours (the solution was changed every 24 hours), the fish were anesthetized with tricaine and placed under a stereo microscope for photography. ImageProPlus was used to count the melanin content at a fixed position of each fish, and the whitening effect of the samples was evaluated by the statistical analysis results of this indicator. The whitening effect of the samples before and after the liposomes was significantly improved. The comparison results are as follows Figure 4 and Figure 5 As shown. Through the zebrafish whitening model, the whitening effect of peony extract liposomes before and after was compared. The results showed that the whitening effect of peony extract liposomes was significantly higher than that of the extract.

[0095] The invention provides a preparation process of peony root bark extract liposomes, which is prepared by a double emulsion method, and the liposome preparation process is optimized with encapsulation rate and stability as indicators, thereby simplifying the liposome preparation process and avoiding the introduction of organic solvents.

[0096] The above description is only a preferred example of the present invention and is not intended to limit the present invention. Although the present invention is described in detail with reference to the aforementioned embodiments, those skilled in the art can still modify the technical solutions described in the aforementioned embodiments or replace some of the technical features therein by equivalents. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A process for preparing liposomes of peony root bark extract, characterized in that: The following steps are involved: S1. Dissolve 0.05-0.2 parts of peony root bark extract in water by mass percentage, and keep warm at 70° C. to obtain a mixed solution phase A; S2, adding 0.5-2.5 parts of PC-80, 0.1-0.5 parts of cholesterol, and 0.2 parts of VE to ethanol, keeping warm at 60°C, and stirring to obtain a mixed solution phase B; S3. During the homogenization of phase B, slowly add phase A; S4, remove ethanol by rotary evaporation, and then add polyols such as pentanediol and glycerol; S5. Obtain peony root bark extract liposomes and store them at low temperature and away from light.

2. The process for preparing liposomes of peony root bark extract according to claim 1, characterized in that: The mass percentage of water in step S1 is 30-70 parts.

3. The process for preparing liposomes of peony root bark extract according to claim 1, characterized in that: The mass percentage of ethanol in step S2 is 30-70 parts.

4. The process for preparing liposomes of peony root bark extract according to claim 1, characterized in that: The ratio of phase A to phase B is 6:4-4:

6.

5. The preparation process of the peony root bark extract liposome according to claim 1, characterized in that: The ratio of peony root bark extract to PC-80 is 1:20-1:

10.

6. The process for preparing liposomes of peony root bark extract according to claim 1, characterized in that: The ratio of PC-80 to cholesterol is 5:

1.

7. The process for preparing liposomes of peony root bark extract according to claim 1, characterized in that: The homogenization conditions of phase B in step S3 are 2000 rpm and 5 min.

8. The process for preparing liposomes of peony root bark extract according to claim 1, characterized in that: In step S4, the amount of pentanediol added is 2-5 parts, and the amount of polyol added is 4-8 parts.

9. The process for preparing liposomes of peony root bark extract according to claim 1, characterized in that: The method for detecting the encapsulation efficiency of the peony root bark extract liposome comprises the following steps: S11, accurately pipette 0.2 mL of liposome solution; S12, add an equal amount of 10 mg / mL protamine and mix thoroughly; S13, centrifuge at 4000 rpm / min for 20 min, and take the supernatant to determine the content of peony root bark extract; S14. Finally, the encapsulation efficiency is calculated.

10. The process for preparing liposomes of peony root bark extract according to claim 1, characterized in that: The stability detection method of the peony root bark extract liposome comprises the following steps: S21, accurately pipette the liposome solution; S22, centrifuge at 4000 rpm / min for 20 min, and take the supernatant; S23, measuring the OD value of the liposomes before and after centrifugation, and calculating the difference; S24. Observe the appearance of liposome solutions under different conditions.

Images