Crayfish astaxanthin precursor liposome and preparation method thereof

Abstract

The invention provides a crayfish astaxanthin proliposome and a preparation method thereof, and belongs to the technical field of preparation of astaxanthin nano-liposomes. The preparation method of the astaxanthin proliposome provided by the invention comprises the following steps: (1) taking crayfish shells, fully freeze-drying the crayfish shells, grinding and sieving the crayfish shells to obtain crayfish shell powder; (2) taking the shrimp shell powder, adding absolute ethyl alcohol, extracting astaxanthin, and collecting an astaxanthin solution; (3) adding phospholipid into the astaxanthin solution to obtain an astaxanthin-phospholipid solution; and (4) adding sugar alcohol particles into the astaxanthin-phospholipid solution, and carrying out rotary evaporation to remove the absolute ethyl alcohol so as to obtain the granular astaxanthin precursor liposome. By adopting the method disclosed by the invention, the extraction of the crayfish shell astaxanthin and the linkage preparation of the astaxanthin precursor liposome can be realized, and the stable astaxanthin liposome can be quickly and efficiently prepared.

Description

Technical Field

[0001] The present invention relates to the technical field of the preparation of astaxanthin nano-liposomes, and particularly to a crayfish astaxanthin precursor liposome and a preparation method thereof. Background Art

[0002] Astaxanthin is a kind of carotenoid, and its chemical name is 3,3′-dihydroxy-4,4′-diketo-β,β′-carotene (C 40 H 52 O4). The special long-chain conjugated olefin structure in its molecular structure endows it with the function of efficiently quenching reactive oxygen species. It is by far the strongest natural antioxidant in nature, 10 times that of β-carotene, 200 times that of lutein, and 550 times that of vitamin E. Crayfish shells are good raw materials for extracting astaxanthin, accounting for 30% of the total weight of crayfish. The content of astaxanthin (Astaxanthin, ASX) in them is as high as 80-90 μg / g, far higher than that of prawns and Antarctic krill (34.43, 55.75 μg / g). The patent with the application number 200810236028.X and the publication date of May 6, 2008 discloses a method for extracting astaxanthin from Procambarus clarkii shells. After drying the shells, chloroform is used for extraction, and finally the extraction rate is about 300 mg / kg of shells. However, it does not overcome the problems that astaxanthin is insoluble in water and difficult to apply.

[0003] Free astaxanthin is unstable and easily oxidized. Moreover, astaxanthin is a fat-soluble substance with poor water solubility. The transdermal effect is not good when used directly, and the bioavailability is low. To solve these problems, carriers can be used to load and embed astaxanthin to improve its solubility and physiological activity.

[0004] Liposome is a very promising astaxanthin loading system. It is a liposome vesicle composed of a phospholipid bilayer, with excellent biocompatibility and delivery ability. Loading astaxanthin can significantly improve its solubility, stability and bioavailability, and reduce degradation and oxidation losses. The authorized patent CN202011505988.9 discloses a preparation method of astaxanthin liposomes. Chloroform is used as an organic solvent and phospholipid is used as a membrane material. The thin film hydration method is used to prepare astaxanthin liposomes, with a particle size of 135-155 nm, a potential of -21 to 45 mV, and an encapsulation efficiency of 60-85%. However, limited by bottleneck problems such as production cost and scale, organic reagent residues and storage instability, the application of liposome dosage forms has not been widely promoted.

[0005] Proliposomes are the precursor form of liposomes. They are small dry solid particles formed by lipid materials and drugs adsorbed on water-soluble carriers. After contacting the aqueous phase, the carrier quickly dissolves and the phospholipids swell to form liposomes. They are considered to be an important strategy to break through the bottleneck of liposome applications. Proliposomes have the following advantages over liposomes: (1) they are suitable for large-scale preparation of liposomes in different scenarios, improving application scenarios; (2) they isolate air moisture and protect drug degradation; (3) they solve the problem of instability and cleavage of liposomes during storage; (4) they are convenient for storage and transportation, saving costs. Chinese patent CN 110478379B discloses a proliposome of total biflavonoids from phytoplankton and a preparation method thereof. The total biflavonoids from phytoplankton were encapsulated in a lipid bilayer by thin film dispersion-ultrasound method combined with freeze-drying technology to prepare proliposomes of total biflavonoids from phytoplankton (P-TBESD). The encapsulation rate is high, the surface is rough and the particle size is about 250nm. This process can significantly improve the stability of the proliposome product during long-term storage. However, this method requires the preparation of a liposome suspension in advance, and the preparation of precursor liposomes by mixing with a lyophilization protectant and freeze-drying, which is a bit complicated.

[0006] Therefore, a convenient and efficient method for preparing astaxanthin proliposomes is developed to realize the linked preparation of astaxanthin extraction and astaxanthin proliposomes, simplify the astaxanthin proliposome preparation process, save the production cost of crayfish astaxanthin liposomes, and realize efficient and long-term storage of astaxanthin, which is beneficial to the application of astaxanthin liposomes in production. Summary of the invention

[0007] The purpose of the present invention is to provide a method for preparing astaxanthin proliposomes, so as to realize the extraction of crayfish astaxanthin and the linked preparation of astaxanthin proliposomes, and improve the storage stability of astaxanthin liposomes.

[0008] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0009] The present invention provides a method for preparing crayfish astaxanthin liposomes, comprising the following steps:

[0010] (1) taking crayfish shells, freeze-drying them, grinding them, and sieving them to obtain shrimp shell powder;

[0011] (2) adding anhydrous ethanol to the shrimp shell powder, stirring to extract astaxanthin, and centrifuging to obtain an astaxanthin solution;

[0012] (3) adding phospholipids to the astaxanthin solution to obtain an astaxanthin-phospholipid solution;

[0013] (4) Adding sugar alcohol particles to the astaxanthin-phospholipid solution, and removing anhydrous ethanol by rotary evaporation to obtain astaxanthin proliposomes.

[0014] Preferably, the grinding conditions in step (1) are: grinding for 5 - 10 min at a power of 1000 - 2000 W; the mesh number of the sieve for sieving is 100 - 300 meshes.

[0015] Preferably, the mass - to - volume ratio of the shrimp shell powder to absolute ethanol in step (2) is 1 g:5 - 20 mL.

[0016] Preferably, the extraction conditions in step (2) are: extraction at 3 - 5 °C in the dark; extraction is carried out 2 - 3 times, and the extraction time for each time is 12 - 24 h.

[0017] Preferably, the mass - to - volume ratio of the phospholipid to the astaxanthin solution in step (3) is 1 g:25 - 100 mL; the phospholipid is soybean phospholipid, sunflower phospholipid, egg phospholipid, shrimp phospholipid or hydrogenated phospholipid, and the phosphatidylcholine content in the phospholipid > 50%.

[0018] Preferably, the sugar alcohol particles in step (4) include one or more of sorbitol, xylitol, mannitol or galactitol, and are in solid particle form at room temperature.

[0019] Preferably, the method for removing absolute ethanol in step (4) is rotary evaporation, and the conditions for rotary evaporation are: rotation speed is 50 - 200 rpm, temperature is 50 - 60 °C, and vacuum degree is - 0.05 - - 0.1 MPa.

[0020] Preferably, it further includes adding the astaxanthin precursor liposome to the aqueous phase and spontaneously hydrating to obtain astaxanthin liposome.

[0021] Preferably, the weight - to - volume ratio of the astaxanthin precursor liposome to pure water is 1 g:5 - 100 mL; the astaxanthin liposome is a single - layer or multi - layer hollow vesicle structure formed by a phospholipid bilayer.

[0022] Beneficial effects

[0023] 1. The present invention improves the comprehensive utilization value of waste shrimp shells of crayfish.

[0024] 2. The present invention realizes the linked production of the extraction of astaxanthin from crayfish shrimp shells and the preparation of astaxanthin precursor liposomes, and improves the production efficiency of astaxanthin liposomes.

[0025] 3. The astaxanthin precursor liposome prepared by the present invention is convenient for storage and transportation, and the real - time preparation of astaxanthin liposome can be realized.

[0026] 4. The astaxanthin precursor liposome prepared by the present invention is a precursor form of astaxanthin liposome, which can isolate moisture and oxygen, has good storage stability, and can effectively protect the degradation of astaxanthin during storage. Brief description of the drawings

[0027] Figure 1 It is the technical roadmap of Example 1;

[0028] Figure 2 It is the appearance diagram of freeze-dried shrimp shell powder and ground shrimp shell powder in Example 1;

[0029] Figure 3 It is the appearance diagram of astaxanthin solution extracted with absolute ethanol and astaxanthin-phospholipid solution in Example 1;

[0030] Figure 4 It is the appearance diagram of sorbitol granules and astaxanthin-loaded sorbitol granules (astaxanthin-loaded proliposomes) in Example 2;

[0031] Figure 5 It is the scanning electron microscope image of sorbitol granules and astaxanthin-loaded sorbitol granules (astaxanthin-loaded proliposomes) in Example 2;

[0032] Figure 6 In it, A is the appearance diagram of astaxanthin-loaded liposomes in Example 2; B is the transmission electron microscope image of astaxanthin-loaded liposomes in Example 2; C is the particle size distribution diagram of astaxanthin-loaded liposomes in Example 2. Detailed implementation manners

[0033] The technical solutions provided by the present invention will be described in detail below in conjunction with the embodiments, but they cannot be understood as limiting the protection scope of the present invention.

[0034] Example 1

[0035] A preparation method of crayfish astaxanthin proliposomes includes the following steps (the technical roadmap is as Figure 1 shown):

[0036] (1) Collect crayfish shrimp shells, wash the shrimp shells clean with tap water, remove the attached meat and internal organs on the shrimp shells, and after rinsing clean, fully drain the water. Place them in a freeze dryer (vacuum degree: 6 Pa, temperature: -60 °C; freeze dry for 2 days) to remove water by freeze drying. The freeze-dried crayfish shrimp shells are as Figure 2 shown in the left figure. Use a powder grinder to grind the freeze-dried shrimp shells at a power of 1000 W for 5 minutes, and collect the shrimp shell powder after passing through a 200-mesh sieve (as Figure 2 shown in the right figure);

[0037] (2) Weigh 100 g of the shrimp shell powder, add 1000 mL of absolute ethanol, extract it in the dark at 4 °C for 12 h, centrifuge it at a gravitational acceleration of 5000 g for 20 minutes to obtain an astaxanthin solution. Repeat the extraction and centrifugation operations 3 times until the shrimp shells have no obvious red color. Combine all the astaxanthin solutions and make up the volume to 3000 mL with absolute ethanol (the sample is as Figure 3 shown in the left figure);

[0038] (3) Take 60 g of soybean lecithin (phosphatidylcholine content> 90%), add 3000 mL of astaxanthin solution described in step (2), and stir thoroughly to obtain astaxanthin-phospholipid solution (sample as Figure 2 as shown in the figure on the right);

[0039] (4) 600 g of sorbitol powder particles were added to the astaxanthin-phospholipid solution in step (3), and after being fully stirred, the solution was placed in a round-bottom rotary evaporator, and the anhydrous ethanol was removed by rotary evaporation in a water bath (temperature of 55° C., speed of 100 rpm, vacuum degree of -0.1 MPa), to obtain astaxanthin proliposomes (sample as Figure 3 As shown in the figure on the right);

[0040] (5) Take 50 g of the astaxanthin proliposomes prepared in step (4), add it to 500 mL of purified water (or other aqueous phases, such as beverages, buffer solutions, electrolyte solutions, and digestive fluids), and stir to obtain astaxanthin liposomes.

[0041] Example 2

[0042] The surface morphology of astaxanthin proliposomes and sorbitol particles in Example 1 was observed using a thermal field scanning electron microscope (SEM). A small amount of particle sample was stuck in a conductive tape, the excess sample was blown off, and the sample was placed on a sample stage for gold spraying. The gold-sprayed sample was placed under SEM for observation, with the voltage set to 12Kv, the current set to 30mA, and the magnification set to 150 and 1000 times. The results are shown in Figure 5 shown.

[0043] The microscopic morphology of the astaxanthin liposomes obtained in Example 1 was observed using a transmission electron microscope (TEM) to verify the liposome structure. The astaxanthin liposomes were diluted with purified water to a phospholipid concentration of 1 mg / mL, stained with a 1% phosphotungstic acid solution for 1 min, and observed under a transmission electron microscope after drying for 4 h; the particle size distribution and potential value of the astaxanthin lipids obtained in Example 1 were measured using a Malvern Nano-Sizer laser nanoparticle size analyzer.

[0044] Depend on Figure 4 It can be seen that both the sorbitol particles and the astaxanthin-loaded sorbitol particles (astaxanthin-loaded proliposomes) are in a uniform particle state, among which the astaxanthin-loaded sorbitol particles are bright orange-red, indicating that the crayfish astaxanthin is successfully combined with sorbitol.

[0045] Depend on Figure 5 It can be seen that there are many fibrous holes on the surface of sorbitol particles, while the surface of astaxanthin-loaded sorbitol particles is smoother, which indicates that the phospholipid and astaxanthin complex is evenly deposited on the surface of sorbitol particles.

[0046] By Figure 6It can be seen that when observing the astaxanthin liposomes obtained in step (5) of Example 1, the appearance results show that the astaxanthin precursor liposomes can form a clear and transparent suspension through simple hydration. Figure 6 A); The scanning electron microscopy result graph shows that the obtained suspension is a multi-layer vesicle liposome structure. Figure 6 B); The particle size distribution result shows that the average particle size of the astaxanthin-loaded nanoliposomes obtained is 97.24 nm, the PDI is 0.18, presenting a uniform single-peak distribution. Figure 6 C), and the surface potential is -34.20 mV.

[0047] In Example 3, the drug loading of astaxanthin in the precursor liposomes in Example 1 and the encapsulation efficiency of astaxanthin in the liposomes obtained by hydration were determined.

[0048] 1. The method for determining the drug loading of astaxanthin in the precursor liposomes is as follows:

[0049] Weigh 100 mg of the precursor liposome particles prepared in step (4) of Example 1, add 2 mL of chloroform, and vortex thoroughly to extract astaxanthin in the particles. Centrifuge at 12000 g for 10 min at 4 °C to separate the chloroform supernatant. Calculate the astaxanthin concentration in the supernatant by comparing with the absorbance standard curve of the astaxanthin-chloroform standard solution (478 nm). The content of astaxanthin in the chloroform is the content of astaxanthin loaded in the precursor liposomes.

[0050] The drug loading of astaxanthin in the precursor liposomes is calculated by the following formula:

[0051] Drug loading = Content of astaxanthin in precursor liposomes / Total weight of precursor liposomes * 100%

[0052] After calculation, the drug loading of astaxanthin in the astaxanthin precursor liposomes in Example (1) is 39.79 μg / g.

[0053] 2. The method for determining the encapsulation efficiency of astaxanthin in the liposomes is as follows:

[0054] Take 100 mg of the astaxanthin precursor liposomes prepared in step (4) of Example 1, add 10 mL of ultrapure water and vortex for hydration to form astaxanthin liposomes. Centrifuge the liposomes at 12000 g for 10 min at 4 °C to remove the unentrapped astaxanthin. Separate the supernatant and add 2 mL of chloroform, and vortex thoroughly to extract astaxanthin in the liposomes. Centrifuge at 5000 g for 10 min at 4 °C to separate the lower chloroform layer. Calculate the astaxanthin concentration in the chloroform layer by comparing with the absorbance standard curve of the astaxanthin-chloroform standard solution (478 nm). The content of astaxanthin in the chloroform is the content of astaxanthin encapsulated in the liposomes.

[0055] The encapsulation efficiency of astaxanthin in the liposomes is calculated by the following formula:

[0056] Entrapment efficiency = Content of astaxanthin entrapped in liposomes / Content of astaxanthin in proliposomes * 100%

[0057] After calculation, the entrapment efficiency of astaxanthin in the astaxanthin liposomes obtained in Example (1) was 93.53%.

[0058] Table 1 summarizes the various parameters of the astaxanthin proliposomes in Example 1 and the liposomes obtained by their hydration.

[0059] Table 1 Analysis of the properties of astaxanthin proliposomes and astaxanthin liposomes obtained by their hydration

[0060]

[0061] In Example 4, the storage stability of astaxanthin in astaxanthin proliposomes was determined.

[0062] The astaxanthin proliposomes prepared in Example 1 were filled with nitrogen protective gas, stored in the dark at 25 °C, and taken out regularly for hydration to prepare liposomes. The particle size change of the formed liposomes and the retention rate of astaxanthin were measured, with newly prepared astaxanthin liposomes stored under the same conditions as the control.

[0063] The retention rate of astaxanthin was calculated by the following formula:

[0064] Retention rate = Content of astaxanthin after storage / Initial content of astaxanthin * 100%

[0065] The content of astaxanthin in the liposomes was determined by referring to the previous method.

[0066] The results of the astaxanthin retention rate are shown in Table 2:

[0067] Table 2 Astaxanthin retention rate (%) of astaxanthin proliposomes after storage and astaxanthin liposomes of the control group after storage (25 °C)

[0068] Storage time (months) 1 2 3 4 5 6 Pro-liposome <![CDATA[97.21±1.07 a > <![CDATA[93.71±1.66 b > <![CDATA[89.12±2.23 c > <![CDATA[83.31±1.01 d > <![CDATA[77.86±2.17 de > <![CDATA[72.63±1.41 e > Liposome <![CDATA[80.48±2.55 A > <![CDATA[64.51±4.30 B > <![CDATA[52.71±1.29 C > <![CDATA[42.94±1.49 D > <![CDATA[34.25±1.85 E > <![CDATA[29.11±1.61 F >

[0069] Data are expressed as "mean ± standard deviation" (n = 3), and different letters in the same row indicate significant differences in samples (p < 0.05).

[0070] As can be seen from Table 2, after 6 months of storage in the dark with gas filling, the retention rate of astaxanthin in astaxanthin proliposomes was 72.63%, while the retention rate of astaxanthin in astaxanthin liposomes was 29.11%, indicating that astaxanthin has a better retention rate in proliposomes.

[0071] The results of the liposome particle size are shown in Table 3:

[0072] Table 3 Particle size data of liposomes formed by hydration of astaxanthin proliposomes after storage and particle size change of astaxanthin liposomes of the control group after storage (nm) (25 °C)

[0073] Storage time (months) 1 2 3 4 5 6 Pro-liposome <![CDATA[91.25±1.73 a > <![CDATA[96.77±1.75 a > <![CDATA[95.30±4.70 a > <![CDATA[93.02±1.88 a > <![CDATA[93.46±2.38 a > <![CDATA[96.29±5.01 a > Liposome <![CDATA[152.14±9.10 A > <![CDATA[175.44±9.77 B > <![CDATA[253.52±20.34 C > <![CDATA[316.26±11.87 D > <![CDATA[523.07±19.23 E > <![CDATA[627.08±24.06 F >

[0074] Data are expressed as "mean ± standard deviation" (n = 3), and different letters in the same row indicate significant differences in samples (p < 0.05).

[0075] As can be seen from Table 3, after 6 months of storage in an inflated and light - protected environment, the astaxanthin - precursor liposomes still retained good ability to form nano - liposomes. The particle sizes of the nano - liposomes obtained after spontaneous hydration were all in the range of 90 - 110 nm. However, the particle size of the astaxanthin liposomes gradually increased during storage, which was caused by liposome oxidation and vesicle aggregation, indicating that the astaxanthin - precursor liposomes have better stability than astaxanthin liposomes.

[0076] The above - mentioned are only the preferred embodiments of the present invention. It should be pointed out that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and modifications can be made, and these improvements and modifications should also be regarded as the protection scope of the present invention.

Claims

1. A preparation method of a crayfish astaxanthin precursor liposome, characterized in that, It includes the following steps: (1) Take crayfish shells, freeze-dry them, grind and sieve to obtain shell powder; (2) Add the obtained shell powder to absolute ethanol, stir to extract astaxanthin, and centrifuge to obtain an astaxanthin solution; (3) Add phospholipids to the astaxanthin solution to obtain an astaxanthin-phospholipid solution; (4) Add sugar alcohol particles to the astaxanthin-phospholipid solution, and rotary evaporate to remove absolute ethanol to obtain astaxanthin precursor liposomes.

2. The preparation method according to claim 1, characterized in that, The grinding conditions in step (1) are: at a power of 1000 - 2000W, grind for 5 - 10min; the mesh number of the sieve for sieving is 100 - 300 meshes.

3. The preparation method according to claim 1, characterized in that, The mass-volume ratio of the shell powder to absolute ethanol in step (2) is 1g:5 - 20mL.

4. The preparation method according to claim 1, characterized in that, The extraction conditions in step (2) are: extract in the dark at 3 - 5°C; extract 2 - 3 times, and the extraction time for each time is 12 - 24h.

5. The preparation method according to claim 1, characterized in that, The mass-volume ratio of phospholipids to the astaxanthin solution in step (3) is 1g:25 - 100mL; the phospholipids are soy phospholipids, sunflower phospholipids, egg phospholipids, shrimp phospholipids or hydrogenated phospholipids, and the phosphatidylcholine content in the phospholipids > 50%.

6. The preparation method according to claim 1, characterized in that, The sugar alcohol particles in step (4) include one or more of sorbitol, xylitol, mannitol or galactitol, and present solid particle form at room temperature.

7. The preparation method according to claim 1, characterized in that, The method for removing absolute ethanol in step (4) is rotary evaporation, and the conditions for rotary evaporation are: the rotation speed is 50 - 200rpm, the temperature is 50 - 60°C, and the vacuum degree is -0.05 - -0.1MPa.

8. The preparation method according to claim 1, wherein, It also includes adding the obtained astaxanthin precursor liposomes to the aqueous phase and spontaneously hydrating to obtain astaxanthin liposomes.

9. The preparation method according to claim 8, wherein, The weight-volume ratio of the astaxanthin precursor liposomes to the aqueous phase is 1g:5 - 100mL; the astaxanthin liposomes are single-layer or multi-layer hollow vesicle structures formed by phospholipid bilayers.

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