Method for purifying (-)-α-bisabolol from fermentation source and preparing nano-liposome

Abstract

The present invention discloses a method for purifying (-)-α-bisabolol from a fermentation source and preparing nano-liposomes thereof, belonging to the field of separation and purification in bio-chemical industry. In the present invention, the (-)-α-bisabolol fermentation broth is subjected to membrane separation concentration, and then based on the characteristics of (-)-α-bisabolol, a separation technology combining resin adsorption, SFE (supercritical fluid extraction) and SFC (supercritical fluid chromatography) is adopted to rapidly prepare high-purity (-)-α-bisabolol. Subsequently, the SFI (supercritical fluid injection method) technology is used to obtain nano-liposomes. The whole method avoids the extraction process, greatly reduces the use of organic solvents, reduces the risk of solvent residues and production costs. By means of the SFI technology, the deficiencies of the existing inclusion technology such as low encapsulation efficiency and cumbersome operation process are solved, and (-)-α-bisabolol nano-liposomes with uniform particle size are rapidly and simply prepared, which can improve its water solubility and permeability and broaden the application range of bisabolol.

Description

Technical Field

[0001] The present invention belongs to the field of biochemistry separation and purification, and particularly relates to a method for purifying and preparing nano-liposomes from fermented (-)-α-bisabolol. Background Art

[0002] (-)-α-bisabolol is a light yellow or colorless viscous liquid with a slightly sweet smell, hardly soluble in water, and easily soluble in organic solvents such as methanol and ethanol. (-)-α-bisabolol has activities such as reducing skin inflammation, antibacterial and anti-irritant, promoting blood circulation to remove blood stasis and relieve pain, and has been widely used in the pharmaceutical and cosmetic industries.

[0003] At present, the methods for producing bisabolol mainly include plant extraction method, chemical synthesis and biosynthesis method. Among them, the plant extraction method mainly separates and purifies plants such as chamomile and German chamomile. This method is easily restricted by factors such as the planting cycle of raw materials, yield, transportation and storage costs, and it is difficult to carry out large-scale industrial production. Chemical synthesis mainly uses farnesol as a raw material, and uses organic solvents and strong acids and other reagents for chemical reactions, and then obtains (-)-α-bisabolol through purification and separation. This method requires a large amount of hazardous chemicals, causes great pollution to the environment, and (-)-α-bisabolol is prone to residual organic solvents and raw materials such as farnesol, which is irritating to the skin. The biosynthesis method has become the main production route at the present stage due to its low cost, low-cost and easily available raw materials, and high optical purity. Patent CN110016458A discloses a method for fermenting (-)-α-bisabolol by microorganisms, with a yield of up to 4.15 g / L. The purification of (-)-α-bisabolol from the fermentation broth generally adopts processes such as extraction, concentration, and molecular distillation with organic solvents such as alkanes, lipids, and alcohols. This method requires the use of a large amount of organic solvents, has high toxicity, and there is a risk of solvent residue.

[0004] The poor water solubility and volatile characteristics of (-)-α-bisabolol limit its local application on the skin. Patent CN118005529A uses the deep eutectic solvent technology to prepare panthenol, ethoxydiglycol and bisabolol into supramolecules, reducing the penetration resistance of active molecules and improving transdermal absorption. However, the whole process requires high-pressure nitrogen protection, and at the same time, it pauses for 2 s every 2 s of ultrasonic treatment, and this alternating treatment lasts for 1 h. The whole process is relatively complex and difficult to realize industrial production. Summary of the Invention

[0005] To solve the problems in the prior art, the present invention proposes a method for purifying fermented (-)-α-bisabolol and preparing (-)-α-bisabolol nano-liposomes. The present invention avoids the use of extraction methods, greatly reducing the amount of organic solvents used and obtaining high-purity (-)-α-bisabolol. At the same time, based on the supercritical fluid injection method (SFI), (-)-α-bisabolol nano-liposomes are simply prepared, with a high encapsulation rate, improved permeability, and good anti-inflammatory activity, which helps to broaden its application scope.

[0006] The method for purifying fermented (-)-α-bisabolol and preparing (-)-α-bisabolol nano-liposomes according to the present invention comprises the following steps: S1. Concentrate the (-)-α-bisabolol fermentation broth obtained by the biological fermentation method by membrane separation, add an adsorption resin to the concentrated solution, and filter to obtain the resin adsorbed with (-)-α-bisabolol;

[0007] S2. Place the resin adsorbed with (-)-α-bisabolol obtained in step S1 in a supercritical fluid extraction tank, and perform supercritical extraction with supercritical CO2 as the extractant and alcohol as the entrainer;

[0008] S3. Purify the extract obtained in step S2 by supercritical fluid chromatography to obtain high-purity (-)-α-bisabolol;

[0009] S4. Dissolve the liposomes and the (-)-α-bisabolol obtained in S3 in a supercritical CO2 / ethanol mixed system to form a homogeneous supercritical phase, and then inject it into PBS buffer through a microfluidic nozzle. The instantaneous expansion of CO2 causes self-assembly of lipids to form monodisperse liposomes. Add a freeze-drying protectant and freeze-dry to obtain (-)-α-bisabolol nano-liposomes.

[0010] In order to remove hyphae and concentrate the target components, in S1, ceramic membranes and nanofiltration membranes are preferably used for membrane separation, and the concentration of (-)-α-bisabolol in the concentrated solution obtained by membrane separation is 10-40 g / L. More preferably, the (-)-α-bisabolol fermentation broth is successively passed through a 50 nm ceramic membrane and a nanofiltration membrane with a molecular weight cut-off of 100 Da to remove hyphae and concentrate, obtaining a concentrated solution of 20-40 g / L.

[0011] According to the preferred embodiment of the present invention, in S1, the addition amount of the adsorption resin is 4-10 times the mass of (-)-α-bisabolol in the concentrated solution; stir during the adsorption process, and adsorb at room temperature for 0.5-2 h; preferably, the addition amount of the adsorption resin is 6-10 times the mass of (-)-α-bisabolol in the concentrated solution; stir during the adsorption process, and adsorb at room temperature for 1-2 h. The adsorption resin is a macroporous adsorption resin, including but not limited to D101, XDA-6 or LX-32.

[0012] According to the preferred embodiment of the present invention, in S2, the extraction pressure of supercritical extraction is 20 - 40 MPa, the extraction temperature is 35 - 55 °C; the entrainer is one of methanol, ethanol, and isopropanol, and the volume ratio of the entrainer is 5 - 30%; the extraction time is 0.5 - 2 h, and the CO2 flow rate is 10 - 30 L / h. More preferably, the extraction pressure is 25 - 30 MPa, the extraction temperature is 40 - 50 °C, the extraction time is 1 - 2 h, the CO2 flow rate is 20 - 30 L / h, and the volume ratio of the entrainer is 10 - 20%.

[0013] According to the preferred embodiment of the present invention, in S3, the chromatographic column in supercritical fluid chromatography is one of Torus 1-AA, Torus Diol, and Torus 2-PIC. The mobile phase uses supercritical CO2 and a modifier. The modifier is methanol or ethanol. The mobile phase uses a linear gradient elution program: within 0 - 10 min, the volume ratio of the modifier linearly increases from 5% to 30%; the back pressure is 20 - 40 MPa. More preferably, the linear change rate of the modifier: 0 - 10 min, 5 - 20%; the back pressure is 20 - 30 MPa. High-purity (-)-α-bisabolol is obtained after supercritical fluid chromatography treatment, and the HPLC purity is ≥98%.

[0014] According to the preferred embodiment of the present invention, in S4, the composition of the liposome includes: phosphatidylcholine, cholesterol, and DSPE-PEG2000. Among them, in terms of molar ratio, phosphatidylcholine:cholesterol:DSPE-PEG2000 = 60:35:5.

[0015] Furthermore, in S4, in the supercritical CO2 / ethanol mixed system, the volume ratio of CO2 to ethanol is 80:20, the pressure is 15 - 20 MPa, and the temperature is 45 - 55 °C. More preferably, the pressure is 18 - 20 MPa, and the temperature is 50 - 55 °C.

[0016] Furthermore, in S4, the pore diameter of the microfluidic nozzle is 50 μm, and the pH of the PBS buffer solution is 6.8.

[0017] Furthermore, in S4, the drug loading amount of (-)-α-bisabolol in the (-)-α-bisabolol nanoliposome is 5 - 15 wt%.

[0018] Furthermore, in S4, the lyoprotectant is trehalose, and the added mass percentage is 5%.

[0019] Compared with the prior art, the present invention combines resin adsorption, SFE (supercritical fluid extraction), and SFC (supercritical fluid chromatography) separation technologies, avoids the extraction process, greatly reduces the use of organic solvents, rapidly prepares high-purity (-)-α-bisabolol, and reduces the risk of solvent residues and production costs. With the help of SFI (supercritical fluid injection) technology, it solves the deficiencies of the existing inclusion technology such as low encapsulation efficiency (≤70%) and cumbersome operation process, and rapidly and simply prepares (-)-α-bisabolol nanoliposomes with uniform particle size, which can improve its water solubility and permeability and broaden the application range of bisabolol. Description of the Drawings

[0020] Figure 1 It is a flow chart of the purification and preparation of (-)-α-bisabolol nanoliposomes according to the present invention;

[0021] Figure 2 It is a physical picture of high-purity (-)-α-bisabolol obtained by supercritical fluid chromatography in an embodiment of the present invention;

[0022] Figure 3 It is an HPLC analysis chart of (-)-α-bisabolol in an embodiment of the present invention;

[0023] Figure 4 It is a particle size distribution chart of (-)-α-bisabolol nanoliposomes obtained in an embodiment of the present invention;

[0024] Figure 5 It is a chart of the anti-inflammatory activity results in the embodiments of the present invention. Detailed Embodiments

[0025] The present invention will be further described and explained below in conjunction with the specific embodiments. The described embodiments are only examples of the present disclosure and do not delimit the scope of limitation. The technical features of each embodiment of the present invention can be combined correspondingly without conflict.

[0026] The raw material used in the method of the present invention can be a fermentation broth containing (-)-α-bisabolol. Combining the characteristics of the biological fermentation broth and the target component (-)-α-bisabolol, the present invention designs a set of separation steps for efficiently purifying (-)-α-bisabolol from the fermentation broth. This separation step is designed based on the characteristics of (-)-α-bisabolol and can efficiently remove other non-target components, so it can be applied to any fermentation broth containing (-)-α-bisabolol; for example, the fermentation broth obtained by fermenting the (-)-α-bisabolol engineering bacteria constructed by methods such as CN202210618377.8, CN202411037711.6, CN202410854366.9, CN202410657020.X, CN202410557782.2, CN202410403364.8, CN202410128367.5, CN201910333500.X. Preferably, the concentration of (-)-α-bisabolol in the fermentation broth is as high as possible. Among them, a typical (-)-α-bisabolol fermentation broth suitable for the method of the present invention is the fermentation broth obtained by the method disclosed in Patent CN201910333500.X (CN110016458A). Under the method disclosed in this patent application, the yield of (-)-α-bisabolol is as high as 4.15 g / L.

[0027] To verify the effectiveness of the present invention, the (-)-α-bisabolol fermentation broth used in the subsequent examples and comparative examples of the present invention was obtained by the strain and fermentation method disclosed in the invention patent "An Engineering Strain for Fermentation Synthesis of α-Bisabolol and Its Construction Method" of CN110016458A. Its general fermentation method is to inoculate the engineering strain constructed in this literature into LB medium, place it at 200 - 220 rpm, 35 - 38 °C for 10 - 12 h, then transfer it to the fermentation medium at an inoculation amount of 5 - 10%, add 20 - 25% (v / v) of n-dodecane, and then place it at 200 - 220 rpm, 35 - 38 °C for cultivation for 50 - 70 h to obtain a fermentation broth containing a certain concentration of (-)-α-bisabolol.

[0028] Example 1

[0029] The 5 L of (-)-α-bisabolol fermentation broth was successively passed through a 50 nm ceramic membrane and a nanofiltration membrane with a molecular weight cut-off of 100 Da to remove mycelia and concentrate, obtaining a concentrated solution of 30 g / L. In the concentrated solution, D101 resin, which was 7 times the amount of (-)-α-bisabolol, was added, stirred, and adsorbed at room temperature (25 °C) for 1 h, followed by filtration. The resin adsorbed with (-)-α-bisabolol was placed in an SFE extraction tank for extraction. The extraction pressure was 30 MPa, the extraction temperature was 45 °C, the CO2 flow rate was 25 L / h, the entrainer was ethanol, and the volume ratio of the entrainer was 15%. The extract was obtained, dissolved in ethanol, and loaded onto an SFC system for separation. Chromatographic column: Torus 1-AA, the mobile phase used supercritical CO2 and a modifier, the modifier: ethanol, linear gradient elution program: 0 - 10 min, the volume ratio of the modifier increased linearly from 5% to 20%, the back pressure was 25 MPa, and 30 g of colorless liquid (-)-α-bisabolol ( Figure 2 for the physical picture) was prepared, with a yield of 80% and an HPLC purity of 98.8%, see Figure 3 .

[0030] The high-purity (-)-α-bisabolol was mixed with liposomes at a drug loading of 10 wt%. The liposome composition was phosphatidylcholine (PC): cholesterol: DSPE-PEG2000 = 60:35:5 (molar ratio). The mixture was dissolved under supercritical CO2 / ethanol (volume ratio 80:20, pressure 20 MPa, temperature 50 °C) to form a homogeneous supercritical phase, and then injected into PBS solution (pH 6.8) through a 50 μm microfluidic nozzle to self-assemble into monodisperse liposomes. After adding 5 wt% trehalose, it was freeze-dried to obtain (-)-α-bisabolol nanoliposomes. The encapsulation efficiency was measured to be 90.1%, the average particle size was 71 nm, and the PDI was 0.25. Figure 4 for the particle size distribution diagram.

[0031] Example 2

[0032] The 5 L of (-)-α-bisabolol fermentation broth was successively passed through a 50 nm ceramic membrane and a nanofiltration membrane with a molecular weight cut-off of 100 Da to remove mycelia and concentrate, obtaining a concentrated solution of 10 g / L. XDA-6 resin, which was 4 times the amount of (-)-α-bisabolol, was added to the concentrated solution, stirred, and adsorbed at room temperature of 25 °C for 0.5 h, followed by filtration. The resin adsorbed with (-)-α-bisabolol was placed in an SFE extraction tank for extraction. The extraction pressure was 20 MPa, the extraction temperature was 35 °C, the CO2 flow rate was 10 L / h, the entrainer was methanol, and the volume ratio of the entrainer was 5%. The extract was obtained, dissolved in ethanol, and loaded onto an SFC system for separation. Chromatographic column: Torus Diol, the mobile phase used supercritical CO2 and a modifier, the modifier: methanol, linear gradient elution program: 0 - 10 min, the volume ratio of the modifier increased linearly from 5% to 30%, the back pressure was 20 MPa, and 26 g of colorless liquid (-)-α-bisabolol was prepared, with a yield of 69% and an HPLC purity of 98.1%.

[0033] The high-purity (-)-α-bisabolol was mixed with liposomes at a drug loading of wt 5%. The liposome composition was phosphatidylcholine (PC): cholesterol: DSPE-PEG2000 = 60:35:5 (molar ratio). The mixture was dissolved under supercritical CO2 / ethanol (volume ratio 80:20, pressure 15 MPa, temperature 45 °C) to form a homogeneous supercritical phase, and then injected into PBS solution (pH 6.8) through a 50 μm microfluidic nozzle to self-assemble into monodisperse liposomes. After adding 5 wt % trehalose, it was freeze-dried to form (-)-α-bisabolol nano-liposomes. The encapsulation efficiency was measured to be 95.1%, the average particle size was 74 nm, and the PDI was 0.23.

[0034] Example 3

[0035] The 10 L of (-)-α-bisabolol fermentation broth was successively passed through a 50 nm ceramic membrane and a nanofiltration membrane with a molecular weight cut-off of 100 Da to remove mycelia and concentrate, obtaining a concentrated solution of 40 g / L. LX-32 resin, which was 10 times the amount of (-)-α-bisabolol, was added to the concentrated solution, stirred, and adsorbed at room temperature of 25 °C for 2 h, followed by filtration. The resin adsorbed with (-)-α-bisabolol was placed in an SFE extraction tank for extraction. The extraction pressure was 40 MPa, the extraction temperature was 55 °C, the CO2 flow rate was 30 L / h, the entrainer was isopropanol, and the volume ratio of the entrainer was 30%. The extract was obtained, dissolved in ethanol, and loaded onto an SFC system for separation. Chromatographic column: Torus 2-PIC, the mobile phase used supercritical CO2 and a modifier, the modifier: ethanol, linear gradient elution program: 0 - 10 min, the volume ratio of the modifier increased linearly from 5% to 25%, the back pressure was 40 MPa, and 63 g of colorless liquid (-)-α-bisabolol was prepared, with a yield of 84% and an HPLC purity of 97.1%.

[0036] Mix high-purity (-)-α-bisabolol with liposomes at a drug loading of 15 wt%. The composition of the liposomes is phosphatidylcholine (PC): cholesterol: DSPE-PEG2000 = 60:35:5 (molar ratio). The mixture is dissolved under supercritical CO2 / ethanol (volume ratio 80:20, pressure 20 MPa, temperature 55 °C) to form a homogeneous supercritical phase, and then injected into a PBS solution (pH 6.8) through a 50-μm microfluidic nozzle to self-assemble into monodisperse liposomes. After adding 5 wt% trehalose, it is freeze-dried to obtain (-)-α-bisabolol nano-liposomes. The encapsulation efficiency is measured to be 85.1%, the average particle size is 69 nm, and the PDI is 0.27.

[0037] Comparative Example 1

[0038] Sequentially pass 5 L of (-)-α-bisabolol fermentation broth through a 50-nm ceramic membrane and a nanofiltration membrane with a molecular weight cut-off of 100 Da to remove mycelia and concentrate to obtain a concentrate of 25 g / L. The concentrate is directly placed in an SFE extraction tank for extraction. The extraction pressure is 30 MPa, the extraction temperature is 45 °C, the CO2 flow rate is 25 L / h, the entrainer is ethanol, and the volume ratio of the entrainer is 15%. Only 0.85 g of a red viscous extract is obtained. No subsequent SFC purification and SFI liposome preparation are carried out. The results show that without the resin adsorption process, (-)-α-bisabolol in the fermentation broth cannot be well enriched, thus affecting the extraction efficiency of SFE. Obviously, resin adsorption is the key point of the present invention. It should be noted that if less than 4 times the amount of adsorption resin is added to the concentrate of Comparative Example 1, after the same SFE and SFC purification as in Example 1, the yield of (-)-α-bisabolol is less than 50%. This may be because the mass of (-)-α-bisabolol in the concentrate exceeds the maximum adsorption capacity of the resin and cannot be fully enriched, resulting in a low yield. If more than 10 times the amount of adsorption resin is added to the concentrate of Comparative Example 1, after SFE and SFC purification, the purity will be slightly lower than that of Examples 1 to 3, and (-)-α-bisabolol is a light red liquid. The possible reason for this is that the excessive resin can not only fully adsorb (-)-α-bisabolol, but also adsorb too much pigment or other impurities, bringing challenges to the subsequent SFC separation.

[0039] Comparative Example 2

[0040] The 5 L of (-)-α-bisabolol fermentation broth was successively passed through a 50 nm ceramic membrane and a nanofiltration membrane with a molecular weight cut-off of 100 Da to remove mycelia and concentrate, obtaining a concentrated solution of 30 g / L. In the concentrated solution, D101 resin, which was 7 times the amount of (-)-α-bisabolol, was added, stirred, and adsorbed at room temperature of 25 °C for 1 h, followed by filtration. The resin adsorbed with (-)-α-bisabolol was placed in an SFE extraction tank for extraction. The extraction pressure was 30 MPa, the extraction temperature was 45 °C, the CO2 flow rate was 25 L / h, the entrainer was ethanol, and the volume ratio of the entrainer was 35%. The extract was obtained, dissolved in ethanol, and loaded onto an SFC system for separation. Chromatographic column: Torus 1-AA, the mobile phase used supercritical CO2 and a modifier, the modifier: ethanol, linear gradient elution program: 0 - 10 min, the volume ratio of the modifier increased linearly from 5% to 20%, the back pressure was 25 MPa, and 32 g of a light red liquid (-)-α-bisabolol was prepared, with a yield of 85.3%, an HPLC purity of 93.1%. The subsequent SFI preparation process was the same as that in Example 1. After determination, the encapsulation efficiency of the nanoliposomes was 90.1%, the average particle size was 68 nm, and the PDI was 0.27.

[0041] The difference from Example 1 was that during the SFE extraction process, the volume ratio of the entrainer ethanol exceeded 30%. At this time, the extractant was in a sub-supercritical fluid state and its properties were closer to those of a liquid. Therefore, when extracting (-)-α-bisabolol, more medium-polarity impurities or pigments were also extracted, reducing the purity of the (-)-α-bisabolol finished product. If the entrainer for SFE extraction in Comparative Example 2 was changed to water, then after the same SFE and SFC processes as in Example 1, the resulting (-)-α-bisabolol finished product was red. The possible reason was that the polarity of water was greater than that of methanol, ethanol, and isopropanol, and it was easier to extract water-soluble pigments and other substances during extraction, affecting the properties of the finished product. When the entrainer for SFE extraction in Comparative Example 2 was replaced with a non-polar solvent such as n-hexane, almost no finished product was obtained. This was related to the extremely low solubility of (-)-α-bisabolol in n-hexane. Obviously, the type and ratio of the entrainer in SFE were also key factors affecting the entire purification process.

[0042] Comparative Example 3

[0043] 5 L of (-)-α-bisabolol fermentation broth was successively passed through a 50 nm ceramic membrane and a nanofiltration membrane with a molecular weight cut-off of 100 Da to remove mycelia and concentrate, obtaining a concentrated solution of 20 g / L. 7 times the amount of (-)-α-bisabolol of D101 resin was added to the concentrated solution, stirred, adsorbed at room temperature of 25 °C for 1 h, and then filtered. The resin adsorbed with (-)-α-bisabolol was placed in an SFE extraction tank for extraction. The extraction pressure was 30 MPa, the extraction temperature was 45 °C, the CO2 flow rate was 25 L / h, the entrainer was ethanol, and the volume ratio of the entrainer was 15%. The extract was obtained, dissolved in ethanol, and loaded onto an SFC system for separation. Chromatographic column: C18, the mobile phase used supercritical CO2 and a modifier, the modifier: ethanol, linear gradient elution program: 0 - 10 min, the volume ratio of the modifier increased linearly from 5% to 20%, the back pressure was 25 MPa, and 35 g of a red viscous substance was prepared, with a yield of 93.3% and an HPLC purity of 57%. No subsequent liposome preparation was carried out. The difference from the example was that a C18 packing was used for separation during the SFC purification process. The results showed that (-)-α-bisabolol had almost no retention on the C18 column and effective impurity separation could not be achieved. In the present invention, the separation materials Torus1-AA and Torus 2-PIC used in the SFC purification both had conjugated systems in their structures, which could generate π-π interactions with the carbon-carbon double bonds in (-)-α-bisabolol, so that it could be retained in the above materials. Torus Diol was a diol group that could generate polar interactions such as hydrogen bonds with the hydroxyl groups in (-)-α-bisabolol and thus be retained.

[0044] Comparative Example 4

[0045] 5 L of (-)-α-bisabolol fermentation broth was successively passed through a 50 nm ceramic membrane and a nanofiltration membrane with a molecular weight cut-off of 100 Da to remove mycelia and concentrate, obtaining a concentrated solution of 30 g / L. 7 times the amount of (-)-α-bisabolol of D101 resin was added to the concentrated solution, stirred, adsorbed at room temperature of 25 °C for 1 h, and then filtered. The resin adsorbed with (-)-α-bisabolol was placed in an SFE extraction tank for extraction. The extraction pressure was 30 MPa, the extraction temperature was 45 °C, the CO2 flow rate was 25 L / h, the entrainer was ethanol, and the volume ratio of the entrainer was 15%. The extract was obtained, dissolved in ethanol, and loaded onto an SFC system for separation. Chromatographic column: Torus 1-AA, the mobile phase used supercritical CO2 and a modifier, the modifier: ethanol, linear gradient elution program: 0 - 10 min, the volume ratio of the modifier increased linearly from 5% to 20%, the back pressure was 25 MPa, and 29 g of colorless liquid (-)-α-bisabolol was prepared, with a yield of 77% and an HPLC purity of 98.6%.

[0046] Mix high-purity (-)-α-bisabolol with liposomes at a drug loading of 20 wt%, and the remaining operations for preparing liposomes are the same as in Example 1. Then, it is measured that the encapsulation efficiency is 67%, the average particle size is 72 nm, and the PDI is 0.29. Obviously, as the drug loading of (-)-α-bisabolol increases, the encapsulation efficiency decreases significantly, indicating that there are not enough liposomes to self-assemble into nanoparticles with it. On the contrary, when the drug loading is less than 5%, although the encapsulation efficiency may be very high, it is easy to cause an excess of liposomes, which will affect its biological activity.

[0047] Performance Test

[0048] Transdermal experiment: The transdermal experiment was carried out using a Franz diffusion cell. Since (-)-α-bisabolol is poorly soluble in water, according to the general guidance principle of dissolution or release inspection, ethanol was added as a cosolvent to the absorption solution. The high-purity (-)-α-bisabolol and (-)-α-bisabolol nano-liposomes prepared in Example 1 were dissolved in PBS solution (pH 6.8)-absolute ethanol (50:50), and were set as the control group and the experimental group respectively, and it was ensured that the total amount of (-)-α-bisabolol in the two groups of experimental objects was the same, and the PBS solution-absolute ethanol mixture was used as the blank group. According to the pig skin penetration model, the transdermal experiment was carried out on pig skin for 1 h, 2 h, 4 h, and 8 h. After the experiment, for the skin, after removing the residual samples on the skin, the skin was cut into pieces, ground in a homogenizer with ethanol, ultrasonic demulsification was carried out, centrifugation was carried out, and the supernatant was taken, and the content of (-)-α-bisabolol was determined by HPLC. Each group of experiments was repeated 3 times in parallel, and the average value was taken. Table 1 shows the results of the retention amount of (-)-α-bisabolol in the skin. The results show that the penetration effect of the (-)-α-bisabolol nano-liposomes prepared by the method of the present invention is significantly better than that of the (-)-α-bisabolol pure product.

[0049] Table 1. Retention amount of (-)-α-bisabolol in the skin

[0050]

[0051] Anti-inflammatory activity test: An epithelial reconstruction model in which interleukin-1α is released from keratinocytes and treated with UV; the group treated only with UV is used as the control group, and the cells treated with (-)-α-bisabolol pure product and (-)-α-bisabolol nano-liposomes (keeping the absolute amount of (-)-α-bisabolol in the two groups the same) are used as experimental group 1 and experimental group 2. The survival amounts of interleukin-1α in the 3 groups were tested respectively. Figure 5 It is shown as anti-inflammatory activity. The results show that after treatment with (-)-α-bisabolol and its nano-liposomes, the production and release of interleukin-1α in UV-stimulated keratinocytes can be inhibited, and both show good anti-inflammatory activity, meaning that (-)-α-bisabolol does not lose its anti-inflammatory activity due to being prepared into nano-liposomes.

[0052] The above-described embodiments merely represent several implementation manners of the present invention. The description thereof is relatively specific and detailed, but it should not be construed as a limitation to the scope of the patent of the present invention. For those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can still be made, and these all fall within the protection scope of the present invention.

Claims

1. A method for preparing nano-liposomes, characterized in that, Including: S1. Concentrate the (-)-α-bisabolol fermentation broth obtained by biological fermentation method by membrane separation. Add an adsorption resin to the concentrated solution and filter to obtain the resin adsorbed with (-)-α-bisabolol. The addition amount of the adsorption resin is 4 to 10 times the mass of (-)-α-bisabolol in the concentrated solution. S2. Place the resin adsorbed with (-)-α-bisabolol obtained in step S1 into a supercritical fluid extraction tank, and perform supercritical extraction with supercritical CO2 as the extractant and an alcohol as the entrainer. The entrainer is one of methanol, ethanol, and isopropanol, and the volume ratio of the entrainer is 5 to 30%. S3. Purify the extract obtained in step S2 in a supercritical fluid chromatograph to obtain high-purity (-)-α-bisabolol. The chromatographic column used in the supercritical fluid chromatograph is one of Torus 1-AA, Torus Diol, and Torus 2-PIC. S4. Dissolve the liposome and the (-)-α-bisabolol obtained in S3 in a supercritical CO2 / ethanol mixed system to form a homogeneous supercritical phase, and then inject it into a PBS buffer through a microfluidic nozzle. The instantaneous expansion of CO2 causes self-assembly of lipids to form monodisperse liposomes. Add a lyoprotectant and freeze-dry to obtain (-)-α-bisabolol nano-liposomes. The drug loading of (-)-α-bisabolol in the (-)-α-bisabolol nano-liposomes is 5 to 15 wt%. The composition of the liposome includes: phosphatidylcholine, cholesterol, DSPE-PEG2000, where in terms of molar ratio, phosphatidylcholine: cholesterol: DSPE-PEG2000 = 60:35:

5.

2. The method for preparing nano-liposomes according to claim 1, characterized in that, In S1, ceramic membranes and nanofiltration membranes are used for membrane separation, and the concentration of (-)-α-bisabolol in the concentrated solution obtained by membrane separation is 10 to 40 g / L.

3. The method for preparing nanoliposomes according to claim 1, wherein, In S1, the (-)-α-bisabolol fermentation broth sequentially passes through a 50 nm ceramic membrane and a nanofiltration membrane with a cut-off molecular weight of 100 Da to remove mycelia and concentrate to obtain a concentrated solution.

4. The method for preparing nano-liposomes according to claim 1, characterized in that, In S1, stirring is carried out during the adsorption process, and the adsorption is carried out at room temperature for 0.5 to 2 h.

5. The method for preparing nano-liposomes according to claim 1, characterized in that, In S2, the extraction pressure of the supercritical extraction is 20 to 40 MPa, the extraction temperature is 35 to 55 °C; the extraction time is 0.5 to 2 h, and the CO2 flow rate is 10 to 30 L / h.

6. The method for preparing nano-liposomes according to claim 1, wherein In S3, the mobile phase uses supercritical CO2 and a modifier. The modifier is methanol or ethanol. The mobile phase uses a linear gradient elution program: within 0 - 10 min, the volume ratio of the modifier linearly increases from 5% to 30%; the back pressure is 20 to 40 MPa.

7. The method for preparing nano-liposomes according to claim 1, wherein In S4, in the supercritical CO2 / ethanol mixed system, the volume ratio of CO2 to ethanol is 80:20, the pressure is 15 to 20 MPa, and the temperature is 45 to 55 °C.

8. The method for preparing nano-liposomes according to claim 1, wherein In S4, the pore diameter of the microfluidic nozzle is 50 μm, and the pH of the PBS buffer is 6.8.

Images