Method for preparing active plant extract lipidosome by subcritical water method and application of active plant extract lipidosome

Abstract

The invention discloses a method for preparing active plant extract lipidosome by a subcritical water method, which comprises the following steps: S1, weighing soybean lecithin, cholesterol and tween-80 in proportion, then weighing an active plant extract and deionized water, adding the active plant extract and deionized water into a high-pressure reaction kettle together, controlling the mass volume ratio of the active plant extract to the deionized water in addition amount to be 1: (10-30) mg / mL, and stirring uniformly; the reaction temperature is 60-180 DEG C, the reaction pressure is 3.5 Mpa, and the reaction time is 10-50 minutes; and S2, after the reaction is finished, taking out the prepared active plant extract liposome suspension, and carrying out ice-bath ultrasonic treatment to obtain the active plant extract liposome. The method is simple in preparation process and low in time cost, the production efficiency is greatly improved, and the realization of liposome industrialization development is facilitated.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of liposome preparation, in particular to a method for preparing active plant extract liposomes by subcritical water method. BACKGROUND

[0002] Liposomes are spherical vesicles formed by self-assembly and dispersion of phospholipids in aqueous solution, and their structure is similar to the phospholipid bilayer of biological membranes, with the central and layers separated by an aqueous phase. Because the bilayer of liposomes has amphiphilic properties, liposomes can encapsulate both hydrophilic active substances and hydrophobic active substances, and are widely used in the fields of biological medicine, food and cosmetics.

[0003] At present, thin film dispersion is one of the most common methods for preparing liposomes. Soybean lecithin and cholesterol are fully dissolved in an organic solvent, the organic solvent is then dried by a rotary evaporator to form a lipid film, and then a phosphate buffer solution or other aqueous medium is added for hydration. After hydration, liposomes are obtained. The absolute value of the Zeta-potential of the liposomes formed by this method is generally within 0-30 mV, and the stability is poor, so it needs to be improved. The liposomes prepared by the thin film dispersion method are further dispersed uniformly by using an ultrasonic cell disruptor, which is a thin film dispersion-ultrasonic method. The absolute value of the Zeta-potential of the liposome solution obtained by this process is generally between 40-60 mV, and the stability is good.

[0004] Although the thin film dispersion-ultrasonic method has high encapsulation efficiency and low cost in the preparation of liposomes, it has many steps, such as dissolving the film material with an organic solvent, drying the organic solvent, hydrating, and finally ultrasonic treatment to obtain liposomes. The disadvantages are solvent residue and complicated preparation process. Especially in daily experimental application, this method has many steps, high time cost, and the liposomes prepared in batches have poor repeatability and large particle size distribution range.

[0005] In summary, it is necessary to find a liposome preparation method with high preparation efficiency, small and concentrated particle size of the prepared liposomes, clean and green solvent, good batch repeatability and ultrahigh solubility. SUMMARY

[0006] To achieve the above-mentioned purpose, on the one hand, the present application provides a method for preparing active plant extract liposomes by subcritical water method, the steps are as follows: S1. Weigh soybean lecithin, cholesterol and Tween-80 according to the proportion, and weigh active plant extract and deionized water and add them into a high-pressure reaction kettle, the reaction temperature is 60-180 ℃, the reaction pressure is 3.5 Mpa, and the reaction time is 10-50 min; S2. After the reaction is completed, the prepared active plant extract liposome suspension is taken out, and ice bath ultrasonic is performed to obtain the active plant extract liposome.

[0007] Further, in S1, the mass ratio of the soybean lecithin, cholesterol, Tween-80 and the active plant extract is 15:5:10:1.

[0008] Further, in S1, the mass / volume ratio of the active plant extract and the added amount of deionized water is 1:10~30 mg / mL.

[0009] Further, in S2, ice bath ultrasonic is performed for 12 min under the condition of 144 W.

[0010] On the other hand, the application also provides an application of the active plant extract liposome prepared by the above method in preparing a liposome hydrogel.

[0011] The above technical solution has the following beneficial effects: The scheme of the application proves the feasibility of preparing the liposome by the subcritical water method, gets rid of the existence of the organic solvent in the liposome system, and truly realizes the green and clean production. Moreover, the preparation process is simple, the time cost is low, the production efficiency is greatly improved, and the industrialization development of the liposome is facilitated. BRIEF DESCRIPTION OF DRAWINGS

[0012] Figure 1 Appearance of 6'-O-caffeoyl arbutin, chlorogenic acid, curcumin and quercetin liposomes prepared by the film dispersion method and the subcritical water method; Figure 2 Particle size and PDI (n=3) of the blank, 6'-O-caffeoyl arbutin, chlorogenic acid, curcumin and quercetin liposomes prepared by the film dispersion method; Figure 3 Particle size and PDI (n=3) of the blank, 6'-O-caffeoyl arbutin, chlorogenic acid, curcumin and quercetin liposomes prepared by the film dispersion method; Figure 4 Influence of reaction time on the appearance of arbutin liposomes; Figure 5 Influence of reaction time on the encapsulation efficiency of arbutin liposomes; Figure 6 Influence of reaction temperature on the appearance of arbutin liposomes; Figure 7 Influence of reaction temperature on the encapsulation efficiency of arbutin liposomes; Figure 8 Influence of water addition amount on the appearance of arbutin liposomes; Figure 9The effect of water addition on the encapsulation efficiency of arbutin liposomes; Figure 10 The biological cryogenic scanning electron micrograph of arbutin liposomes prepared by subcritical water method. DETAILED DESCRIPTION

[0013] To illustrate the technical content, structural features, purposes and effects of the technical solutions in detail, the following will be described in detail in conjunction with specific embodiments and the accompanying drawings. Example 1

[0014] A method for preparing active plant extract liposomes by subcritical water method, comprising the following steps: S1. Accurately weigh 0.0300 g of soybean lecithin, 0.0100 g of cholesterol and 0.0200 g of Tween-80 in a ratio of 3:1:2, and then weigh 0.0020 g of active plant extract and 40 mL of deionized water (the mass-volume ratio of the addition amount of active plant extract to deionized water is 1:20 mg / mL) together into a high-pressure reaction kettle, where the active plant extract is arbutin; the reaction temperature is 60°C, the reaction pressure is 3.5 Mpa, and the reaction time is 10 min.

[0015] S2. After the reaction is completed, the prepared arbutin liposome suspension is taken out, and ice bath ultrasonic treatment is performed at 144 W for 12 min (2s / 2s) to obtain arbutin liposomes.

[0016] Comparative Examples 1-8 Different from Example 1, Comparative Examples 1-8 are 6'-O-caffeoyl arbutin, chlorogenic acid, curcumin, and quercetin liposomes obtained by different preparation methods, specifically, the above four kinds of active plant extract liposomes are prepared by using the film dispersion-ultrasonic method and the subcritical method, respectively.

[0017] The particle size distribution and Zeta-potential are measured by using a nanoparticle size analyzer, Figure 1 、 Figure 2 and Figure 3 The effects of two preparation methods on the particle size and Zeta-potential of liposomes (n=3). Combined with Figures 1-3As shown, compared with the various types of drug-encapsulated liposomes prepared by the thin film dispersion-ultrasonic method, the liposomes prepared by the subcritical method are more transparent and uniform, without obvious milky white. It was found during preparation that the liposomes prepared by the subcritical water method appeared milky white before ultrasonic, but became more transparent after ultrasonic. This may be because the liposomes prepared by the subcritical water method have smaller particle size. It was determined that, under the same ratio, the particle size of the liposomes prepared by the thin film dispersion method was about 300 nm, while the particle size of the liposomes prepared by the subcritical water method was about 100 nm, and the particle size distribution was more concentrated, so the liposomes became more transparent after ultrasonic. At the same time, as can be seen from the figure, under the same ratio, the particle size of the liposomes prepared by the subcritical water method is smaller and the distribution is more concentrated. The particle size of the blank, 6'-O-caffeoyl arbutin, chlorogenic acid, curcumin and quercetin liposomes prepared by the subcritical water method is maintained within the range of 100±10 nm, and the PDI is always within 0.3, indicating that the liposome distribution is relatively concentrated. Under the same ratio, the particle size of the various types of liposomes prepared by the thin film dispersion method is distributed within the range of 250-350 nm, and the PDI exceeds 0.3, and the distribution is relatively dispersed.

[0018] Examples 2-5 Different from Example 1, Examples 2-5 prepared arbutin liposomes with reaction time of 20 min, 30 min, 40 min and 50 min, respectively.

[0019] The encapsulation efficiency was measured by UV, and the particle size distribution and Zeta potential were measured by a nanoparticle size analyzer. Table 1 shows the effect of reaction time on the particle size and Zeta potential of arbutin liposomes (n=3)

[0020] Combining Figures 4-5 As shown, with the increase of reaction time, the encapsulation efficiency of arbutin liposomes showed a trend of first increasing and then decreasing, reaching a peak of 68.5% when the reaction time was 30 min. At this time, the particle size of the liposomes was 106.3 nm, the PDI was 0.275, and the Zeta potential was -54.32 mV. This is because the reaction time is short, and the liposomes are not fully formed, which reduces the encapsulation efficiency. When the reaction time is within 10-30 min, the particle size and PDI of the arbutin liposomes are relatively stable, always within the range of 105±18 nm and 0.28±0.05 mV, with little change. With the increase of reaction time, the Zeta potential of the arbutin liposomes always maintains within the range of -45±10 mV, with an absolute value always >30 mV, the liposomes are in a stable state, and the prepared arbutin liposome solution is uniform and transparent. It shows that the reaction time has a certain effect on the encapsulation efficiency of arbutin liposomes, but has little effect on the particle size, PDI and Zeta potential.

[0021] Examples 6-9 Unlike Example 1, Examples 6-9 prepared arbutin liposomes at reaction temperatures of 90 °C, 120 °C, 150 °C, and 180 °C, respectively.

[0022] The encapsulation efficiency was measured using ultraviolet light, and the particle size distribution and zeta potential were measured using a nanoparticle size analyzer. Table 2 shows the effect of reaction temperature on the particle size and zeta potential of arbutin liposomes (n=3).

[0023] Combination Figures 6-7 As shown, with the continuous increase of reaction temperature, the liposome encapsulation initially increased slowly to 67.9%, then plummeted to 19%, and the solution color changed from uniform and clear to pale yellow. The particle size also decreased from 105.7 nm to 82.05 nm, and the Zeta-potential increased from -48.02 mV to -29.01 mV, with an absolute value <30 mV, indicating that the liposomes were unstable to some extent. Within the reaction temperature range of 60-150 ℃, the particle size, PDI, and Zeta-potential all met the experimental requirements, and the solution remained uniform and clear, indicating good liposome stability. Analysis revealed that these results were caused by the high-temperature oxidation of phospholipids in the liposome membrane material, leading to solution yellowing, liposome breakage, drug leakage, a sharp drop in encapsulation efficiency, and a Zeta-potential absolute value <30 mV.

[0024] Examples 10-13 Unlike Example 1, Examples 10-13 prepared arbutin liposomes with added amounts of 20 mL, 30 mL, 50 mL, and 60 mL of deionized water, respectively, with corresponding mass-to-volume ratios of arbutin to deionized water of 1:10, 1:15, 1:25, and 1:30 mg / mL.

[0025] The encapsulation efficiency was measured using ultraviolet light, and the particle size distribution and zeta potential were measured using a nanoparticle size analyzer. Table 3 shows the effect of water addition on the particle size and zeta potential of arbutin liposomes (n=3).

[0026] like Figure 8 As shown, with the continuous increase of the amount of deionized water added, the encapsulation efficiency of arbutin liposomes showed a trend of rapid increase followed by slow decrease. When the amount of deionized water added was 40 mL, the encapsulation efficiency of arbutin liposomes was the highest at 71.2%.

[0027] like Figure 9As shown, the arbutin liposome solution was uniform and clear, with no obvious aggregation or yellowing, indicating good liposome stability. With the addition of deionized water ranging from 30 to 60 mL, the particle size, PDI, and Zeta-potential remained relatively stable. The particle size remained within the range of 105 ± 10 nm, the PDI remained within the range of 0.28 ± 0.04 mV, and the absolute value of the Zeta-potential was consistently > 30 mV, demonstrating the good stability of the arbutin liposomes.

[0028] When 20 mL of deionized water was added, the arbutin liposomes exhibited the lowest encapsulation efficiency at only 21.5%, with a small particle size of 89.35 nm, a PDI of 0.297, and a Zeta potential of -33.64 mV. This is likely because the amount of deionized water added was too small, resulting in the liposomes remaining almost entirely in a gaseous state during the reaction, thus hindering membrane dissolution and causing a sharp drop in encapsulation efficiency.

[0029] like Figure 10 As shown in the cryo-electron microscopy images, the liposomes prepared using the subcritical water method exhibit a well-defined spherical vesicle structure and a small particle size distribution, generally maintained at around 100 nm, compared to the thin-film dispersion method. In summary, the subcritical water method of this application can prepare a series of liposomes with small particle size, concentrated distribution, and good stability. Furthermore, the liposome solution is more homogeneous and transparent, without obvious milky white color or precipitation. Example 14

[0030] The preparation steps of an arbutin liposome gel are as follows: Accurately weigh 1 g of carbomer 980 powder and add it to 100 mL of deionized water. Stir at room temperature for 24 hours to allow it to fully swell. Mix the prepared carbomer matrix with an equal volume of arbutin liposomes prepared in Example 1 and chitosan aqueous solution, continue stirring, and add dropwise a 50% (w / v) triethanolamine crosslinking agent to form a gel. The resulting liposome hydrogel is uniform and transparent, without precipitation, and meets relevant standard requirements, making it suitable for further development into novel cosmetic applications.

[0031] It should be noted that, in this document, relational terms such as "first" and "second" are used only to distinguish one entity or operation from another, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Furthermore, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or terminal device that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or terminal device. Unless otherwise specified, an element defined by the phrase "comprising..." or "including..." does not exclude the presence of additional elements in the process, method, article, or terminal device that includes said element. Additionally, in this document, "greater than," "less than," "exceeding," etc., are understood to exclude the stated number; "above," "below," "within," etc., are understood to include the stated number.

[0032] Although the above embodiments have been described, those skilled in the art, once they understand the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the above descriptions are merely embodiments of the present invention and do not limit the scope of patent protection of the present invention. Any equivalent structural or procedural transformations made using the content of the present invention's specification and drawings, or direct or indirect applications in other related technical fields, are similarly included within the scope of patent protection of the present invention.

Claims

1. A method for preparing active plant extract liposomes using subcritical water, characterized in that, The steps are as follows: S1. Weigh soybean lecithin, cholesterol and Tween-80 according to the proportion, then weigh active plant extract and deionized water and add them together into the high-pressure reactor. The reaction temperature is 60-180℃, the reaction pressure is 3.5 MPa, and the reaction time is 10-50 min. S2. After the reaction is complete, take out the prepared active plant extract liposome suspension and sonicate it in an ice bath to obtain active plant extract liposomes.

2. The method as described in claim 1, characterized in that, In S1, the mass ratio of soybean lecithin, cholesterol, Tween-80 and active plant extract is 15:5:10:

1.

3. The method as described in claim 2, characterized in that, In S1, the mass-to-volume ratio of the active extract to deionized water is 1:10~30 mg / mL.

4. The method as described in claim 1, characterized in that, In S2, the ice bath was sonicated for 12 minutes at 144 W.

5. The application of an active plant extract liposome prepared by any one of the methods described in claims 1-4 in the preparation of liposome hydrogels.

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