CN107753433B - Nano cordyceps militaris peptide liposome, preparation method thereof and oral liquid preparation - Google Patents
Nano cordyceps militaris peptide liposome, preparation method thereof and oral liquid preparation Download PDFInfo
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Abstract
The invention relates to a nano cordyceps militaris peptide liposome, a preparation method thereof and an oral liquid preparation, belonging to the technical field of liposome preparation. The invention provides a nano cordyceps militaris peptide liposome which comprises nano cordyceps militaris peptide and a liposome membrane layer, wherein the nano cordyceps militaris peptide is wrapped in the liposome membrane layer, and the feeding materials of the liposome membrane layer comprise the following components in parts by weight: 6-7 parts of soybean hydrogenated lecithin, 6-9 parts of cholesterol, 17-18 parts of normal hexane, 18-19 parts of chloroform and 9-10 parts of water; the liposome membrane layer comprises the raw materials of soybean hydrogenated lecithin and cholesterol. The invention provides a nano cordyceps militaris peptide liposome and a preparation method thereof. The nano cordyceps militaris peptide liposome provided by the invention has the advantages of high encapsulation rate, good cell compatibility and simple production process.
Description
Technical Field
The invention relates to the technical field of liposome preparation, and in particular relates to a nano cordyceps militaris peptide liposome, a preparation method thereof and an oral liquid preparation.
Background
Cordyceps militaris peptide has antioxidant and blood sugar lowering activity, and can be used for adjuvant treatment of diabetes. However, the peptide substances are easy to be decomposed due to the special acidic environment in the stomach, and the absorption and utilization of the peptide substances are influenced due to the small absorption area of the gastric mucosa.
The liposome is an artificially prepared closed saccular structure which is spontaneously formed in water by virtue of hydrophobic interaction among phospholipid molecules, and the medicament is wrapped in the liposome. The liposome is simple to prepare, has no toxicity to organisms and no immunogenicity, and is mainly phagocytized by the grid cells after entering the body, so that the utilization rate of the medicine can be improved. The liposome membrane is composed of lecithin and cholesterol, the lecithin is a component of body cells, is a physiologically active substance, has a regulation effect on body functions, can promote metabolism and enhance the resistance of a human body, and the liposome can be prepared into injections, oral preparations or aerosols and the like to realize administration in various ways. Therefore, it is regarded as a novel drug carrier. The distribution of the drug in the tissues can be controlled by changing the size and charge of the liposome or adding certain components, thereby realizing targeted drug delivery.
The conventional material for preparing liposomes is unsaturated lecithin, which is easily oxidized by oxygen in the air due to the double bond, causing membrane rupture and leakage of the drug. Therefore, nitrogen is used as a protective gas to prevent lipid oxidation in the processes of film formation, ultrasonic treatment, filling and storage in the preparation process. Liposomes can be prepared by a variety of methods. The film dispersion method is that lipid such as lecithin, cholesterol and the like and fat-soluble drugs are dissolved in chloroform (or other organic solvents), then rotary evaporation is carried out, so that a film is formed on the inner wall of a bottle, water-soluble drugs are dissolved in phosphate buffer solution and added into the flask to be continuously stirred, and then the macroporous liposome with larger molecular radius is obtained, but the method needs high organic solvent concentration, phosphate needs to be added for dissolving the embedded drugs, the encapsulation rate of the liposome is generally higher, but the permeability is poor; the ultrasonic method is that the aqueous solution of the medicine is mixed with the organic solvent dissolved with lipid and then the mixture is treated by ultrasonic to prepare the liposome, the method is simple and easy to operate, but the prepared liposome has lower entrapment rate; the freeze-drying method is particularly suitable for medicines which are unstable under heat, but the freeze-drying method can reduce the encapsulation efficiency of the liposome preparation; the reverse phase evaporation method is a common method for preparing liposome, the obtained liposome is large unilamellar liposome, the volume is generally large, the method is suitable for wrapping water-soluble drugs and macromolecular bioactive substances, the encapsulation volume is high, the encapsulation efficiency is low, and the particle size of the liposome molecules obtained by the method is larger than 100 nm. The prior art can not obtain a high-efficiency and stable cordyceps militaris peptide liposome.
Disclosure of Invention
The invention aims to provide a nano cordyceps militaris peptide liposome, a preparation method thereof and an oral liquid preparation. The nano cordyceps militaris peptide liposome provided by the invention has the advantages of high encapsulation rate, good cell compatibility, high stability and simple production process.
The invention provides a nano cordyceps militaris peptide liposome which comprises nano cordyceps militaris peptide and a liposome membrane layer, wherein the nano cordyceps militaris peptide is wrapped in the liposome membrane layer, and the feeding materials of the liposome membrane layer comprise the following components in parts by weight: 6-7 parts of soybean hydrogenated lecithin, 6-9 parts of cholesterol, 17-18 parts of normal hexane, 18-19 parts of chloroform and 9-10 parts of water;
the liposome membrane layer comprises the raw materials of soybean hydrogenated lecithin and cholesterol.
The invention also provides a preparation method of the nano cordyceps militaris peptide liposome, which comprises the following steps:
1) mixing the hydrogenated soybean lecithin with cholesterol to obtain a solid mixture;
2) mixing normal hexane and chloroform to obtain a mixed solvent;
3) mixing and dissolving the solid mixture obtained in the step 1) and the mixed solvent obtained in the step 2) to obtain a mixed solution;
4) removing the organic solvent in the mixed solution in the step 3) to obtain a film;
5) mixing the film obtained in the step 4) with the nano cordyceps militaris peptide, hydrating for 35-45 min at 45-50 ℃, and carrying out ultrasonic cell crushing treatment for 4.5-5 min to obtain hydrated liposome;
6) freezing and centrifuging the hydrated liposome obtained in the step 5), and freezing and drying the supernatant to obtain the nano cordyceps militaris peptide liposome;
the step 1) and the step 2) are not limited in chronological order.
Preferably, the molecular weight of the nano cordyceps militaris peptide in the step 5) is not more than 5000 daltons; the nano cordyceps militaris peptide is mixed with the film in a form of nano cordyceps militaris peptide solution, and the pH value of the nano cordyceps militaris peptide solution is 6.5-8.0.
Preferably, the preparation method of the nano cordyceps militaris peptide solution comprises the following steps: ball-milling the fruiting body of Cordyceps militaris for 30min to obtain nanometer Cordyceps militaris powder; dissolving the nano cordyceps militaris powder in water, fermenting for 24-36 hours at the pH value of 6.5-8.0 and the temperature of 30-40 ℃, centrifuging for 10-15 min, and performing 5000 dalton molecular weight cut-off ultrafiltration on the obtained supernatant to obtain a filtrate, namely a nano cordyceps militaris peptide solution.
Preferably, the mass-to-water volume ratio of the nano cordyceps militaris powder is 1-3 g: 30-40 mL.
Preferably, the organic solvent removing method in the step 4) comprises the following steps: rotary evaporating at 65-70 deg.c for 20-30 min.
Preferably, the adding mass of the nano cordyceps militaris peptide in the step 5) is 0.5 time of that of the mixed solution in the step 3).
Preferably, the conditions of the freezing and centrifuging in the step 6) are 0-4 ℃ and 3000-4000 r/min.
Preferably, the conditions of the freeze-drying in the step 6) are as follows: the sublimation temperature is 10-15 ℃ and the time is 4-6 hr.
The invention also provides an oral liquid preparation containing the cordyceps militaris peptide liposome or the cordyceps militaris peptide liposome prepared by the preparation method.
The invention provides a nano cordyceps militaris peptide liposome. The nano cordyceps militaris peptide liposome provided by the invention adopts hydrogenated soybean lecithin as a coating material, and the liposome can be prepared under the conventional conditions by adjusting the component proportion of a film-forming material, so that the oxidation reaction is avoided, and the obtained nano cordyceps militaris peptide liposome has the advantages of high encapsulation rate, good cell compatibility, high stability, small molecular particle size, easiness in penetrating cell membranes and easiness in being absorbed by a human body. Test results show that the encapsulation rate of the nano cordyceps militaris peptide liposome provided by the invention is up to 48.14%, the liposome is easy to permeate cell membranes, and the stability of the antioxidant activity of the nano cordyceps militaris peptide can be ensured at the pH value of 7.0 and the temperature of 37 ℃; in addition, the particle size distribution of the liposome obtained by the invention is 57 +/-21 nm, which is smaller than the molecular particle size of the unsaturated lecithin liposome, so that the liposome is easier to permeate cell membranes and is absorbed by human bodies. The particle size of the liposome is less than 100nm, which is more beneficial to the transdermal absorption and the in vivo delivery of the medicament.
Drawings
FIG. 1 is a transmission electron microscope photograph of the nano cordyceps militaris peptide liposome microsphere provided in embodiment 1 of the present invention;
FIG. 2 is a transmission electron microscope photograph of the nano cordyceps militaris peptide liposome solution provided in embodiment 1 of the present invention;
FIG. 3 is a graph showing the effect of the ratio of the amount of hydrogenated soybean lecithin to the amount of cholesterol substance on the encapsulation efficiency, provided in example 4 of the present invention;
FIG. 4 is a graph showing the effect of hydration temperature on encapsulation efficiency provided in example 4 of the present invention;
FIG. 5 is a graph showing the effect of sonication time on encapsulation efficiency provided in example 4 of the present invention;
FIG. 6 is a result chart of the effect of pH of the Cordyceps militaris peptide solution on the encapsulation efficiency provided in embodiment 4 of the present invention;
FIG. 7 is a graph showing the results of trypsin tolerance of liposomes provided in example 5 of the present invention;
FIG. 8 is a graph showing the results of liposome reactions at different temperatures provided in example 6 of the present invention.
Detailed Description
The invention provides a nano cordyceps militaris peptide liposome which comprises nano cordyceps militaris peptide and a liposome membrane layer, wherein the nano cordyceps militaris peptide is wrapped in the liposome membrane layer, and the feeding materials of the liposome membrane layer comprise the following components in parts by weight: 6-7 parts of soybean hydrogenated lecithin, 6-9 parts of cholesterol, 17-18 parts of normal hexane, 18-19 parts of chloroform and 9-10 parts of water;
the liposome membrane layer comprises the raw materials of soybean hydrogenated lecithin and cholesterol.
In the invention, the molecular weight of the nano cordyceps militaris peptide is not more than 5000 daltons; the nano cordyceps militaris peptide is mixed with the film in a form of nano cordyceps militaris peptide solution, and the pH value of the nano cordyceps militaris peptide solution is 6.5-8.0. In the invention, the pH value of the nano cordyceps militaris peptide solution is preferably 6.8. In the invention, the preparation method of the nano cordyceps militaris peptide solution comprises the following steps: ball-milling the fruiting body of Cordyceps militaris for 30min to obtain nanometer Cordyceps militaris powder; dissolving the nano cordyceps militaris powder in water, fermenting for 24-36 hours at the pH value of 6.5-8.0 and the temperature of 30-40 ℃, centrifuging for 10-15 min, and performing 5000 dalton molecular weight cut-off ultrafiltration on the obtained supernatant to obtain a filtrate, namely a nano cordyceps militaris peptide solution. In the invention, the nano cordyceps militaris peptide powder is dissolved in water, and is preferably fermented for 24 hours at the pH value of 6.8 and the temperature of 35 ℃ and then centrifuged for 10 min. In the invention, the mass-to-water volume ratio of the nano cordyceps militaris powder is 1-3 g: 30-40 mL, preferably 1 g: 40 mL.
In the invention, the feeding of the liposome membrane layer comprises 6-7 parts of soybean hydrogenated lecithin, more preferably 6.5 parts, and in the invention, the addition of the soybean hydrogenated lecithin has the effect of enabling the liposome to have a lower phase transition temperature. The source of the hydrogenated soybean lecithin is not particularly limited in the present invention, and a conventional commercial product of hydrogenated soybean lecithin known to those skilled in the art may be used.
In the invention, the feeding of the liposome membrane layer comprises 6-9 parts of cholesterol, and more preferably 8 parts. In the present invention, the cholesterol functions to provide good fluidity and viscosity reduction to the liposome membrane. The source of the cholesterol is not particularly limited in the present invention, and a conventional commercial product of cholesterol well known to those skilled in the art may be used. In the present invention, the mixing of the hydrogenated soybean lecithin and cholesterol in the above-mentioned weight parts can increase the stability of the liposome prepared.
In the invention, the feeding of the liposome membrane layer comprises 17-18 parts of n-hexane, and preferably 17.5 parts. In the present invention, the n-hexane is used to dissolve the hydrogenated soybean lecithin. In the invention, the feeding of the liposome membrane layer comprises 18-19 parts of chloroform, and more preferably 18.5 parts. In the present invention, the chloroform is used to dissolve cholesterol. In the invention, the mixing of the normal hexane and the chloroform according to the weight parts can enhance the mutual solubility uniformity of the soybean hydrogenated lecithin and the cholesterol. The source of the n-hexane and chloroform is not particularly limited in the present invention, and a commercially available product of conventional n-hexane and chloroform known to those skilled in the art may be used.
In the invention, the feeding of the liposome membrane layer comprises 9-10 parts of water, and preferably 9.5 parts of water. The source of the water is not particularly limited, and common distilled water can be adopted. In the present invention, the addition of water is carried out while the liposomes are subjected to a hydration step, said water acting to form a two-phase system.
In the invention, the proportion of the soybean hydrogenated lecithin, the cholesterol, the normal hexane, the chloroform and the water can realize the maximum encapsulation rate of the liposome preparation, and the invention has the advantages of high pharmacological activity, leakage reduction and the like. The particle size distribution of the nano cordyceps militaris peptide liposome prepared by the invention is 57 +/-21 nm, the particle size distribution is smaller than the molecular particle size of the unsaturated lecithin liposome, the nano cordyceps militaris peptide liposome is easier to permeate cell membranes and is absorbed by human bodies, and the particle size of the liposome is smaller than 100nm, so that the nano cordyceps militaris peptide liposome is more favorable for transdermal absorption and in-vivo delivery of medicaments.
The invention also provides a preparation method of the nano cordyceps militaris peptide liposome, which comprises the following steps:
1) mixing the hydrogenated soybean lecithin with cholesterol to obtain a solid mixture;
2) mixing normal hexane and chloroform to obtain a mixed solvent;
3) mixing the solid mixture obtained in the step 1) with the mixed solvent obtained in the step 2) to obtain a mixed solution;
4) removing the organic solvent in the mixed solution in the step 3) to obtain a film;
5) mixing the film obtained in the step 4) with the nano cordyceps militaris peptide, hydrating for 35-45 min at 45-50 ℃, and carrying out ultrasonic cell crushing treatment for 4.5-5 min to obtain hydrated liposome;
6) freezing and centrifuging the hydrated liposome obtained in the step 5), and freezing and drying the supernatant to obtain the nano cordyceps militaris peptide liposome;
the step 1) and the step 2) are not limited in chronological order.
The invention mixes the soybean hydrogenated lecithin and cholesterol to obtain a solid mixture. In the present invention, the mixing weight ratio of the hydrogenated soybean lecithin and cholesterol is preferably 6: 7, and the mixture of the hydrogenated soybean lecithin and cholesterol can increase the fluidity of the liposome membrane.
The invention mixes normal hexane and chloroform to obtain a mixed solvent. In the present invention, the mixing weight ratio of n-hexane and chloroform is preferably 1: 1.
After the solid mixture and the mixed solvent are obtained, the solid mixture and the mixed solvent are mixed and dissolved to obtain the mixed solution. In the present invention, the weight ratio of the solid mixture to the mixed solvent is preferably 1: 3.
After the mixed solution is obtained, the organic solvent in the mixed solution is removed to obtain the film. In the invention, the method for removing the organic solvent comprises the following steps: rotary evaporation at 65-70 deg.C for 20-30 min, preferably at 65 deg.C for 25 min. The stability of the lipid bilayer film formed by the limitation of the temperature and the time is good. The device for rotary evaporation is not particularly limited in the present invention, and a rotary evaporator well known to those skilled in the art may be used.
After the film is obtained, the film and the nano cordyceps militaris peptide are mixed, hydration is carried out for 35-45 min at the temperature of 45-50 ℃, and ultrasonic cell crushing treatment is carried out for 4.5-5 min, so as to obtain hydrated liposome. In the invention, the nano cordyceps militaris peptide is a nano cordyceps militaris peptide solution with the pH value of 6.5-8.0, and more preferably 6.8. In the invention, the preparation method of the nano cordyceps militaris peptide solution comprises the following steps: ball-milling the fruiting body of Cordyceps militaris for 30min to obtain nanometer Cordyceps militaris powder; dissolving nano cordyceps militaris powder in water, fermenting at the pH of 6.5-8.0 and the temperature of 30-40 ℃ for 24-36 hours, centrifuging for 10-15 min, taking supernatant, performing 5000 dalton molecular weight cut-off ultrafiltration, and taking filtrate as nano cordyceps militaris peptide solution. In the invention, the nano cordyceps militaris peptide powder is dissolved in water, and is preferably fermented for 24 hours at the pH value of 6.8 and the temperature of 35 ℃ and then centrifuged for 10 min.
In the invention, the mass-to-water volume ratio of the nano cordyceps militaris powder is 1-3 g: 30-40 mL, preferably 1 g: 40 mL. In the invention, the adding mass of the nano cordyceps militaris peptide is preferably 0.5 time of the mass of the mixed solution. In the invention, the content of the nano cordyceps militaris peptide in each 5mg of liposome is preferably 500-1500 mug. In the invention, the hydration temperature is preferably 50 ℃, and the setting of the temperature improves the phase transition temperature of hydration film forming, so that the encapsulation efficiency and the stability of the liposome are improved.
After obtaining the hydrated liposome, the hydrated liposome is subjected to freeze centrifugation, and the supernatant is subjected to freeze drying to obtain the nano cordyceps militaris peptide liposome, wherein the conditions of the freeze centrifugation are 0-4 ℃ and 3000-4000 r/min. In the present invention, the conditions of the freeze-drying are: the sublimation temperature is 10-15 ℃ and the time is 4-6 hr. In the invention, the freeze-drying condition can increase the stability of the antioxidant activity of the liposome-coated nano cordyceps militaris peptide, and the adverse effect of freeze-drying on the encapsulation efficiency can be solved by combining the addition of the soybean hydrogenated lecithin in the preparation of the liposome material, so that the encapsulation efficiency of the obtained liposome is high.
The invention adopts a rotary film-ultrasonic method, and simplifies the preparation process of the liposome. The conventional material for preparing the liposome is lecithin containing unsaturated bonds, and a protection device is required to prevent lipid oxidation in the links of film formation, ultrasonic treatment, filling and storage in the preparation process. The particle size distribution of the nano cordyceps militaris peptide liposome prepared by the invention is 57 +/-21 nm, is smaller than the molecular particle size of the unsaturated lecithin liposome, and is easier to permeate cell membranes and absorb by human bodies. The particle size of the liposome is less than 100nm, which is more beneficial to the transdermal absorption and the in vivo delivery of the medicament.
The invention also provides an oral liquid preparation containing the cordyceps militaris peptide liposome or the cordyceps militaris peptide liposome prepared by the preparation method. The nanometer cordyceps militaris peptide liposome provided by the invention has good stability, can avoid degradation of protease, can be used for preparing high-efficiency oral liquid preparations, and the hypoglycemic and antioxidant activities of the obtained nanometer cordyceps militaris peptide liposome are consistent with those of uncoated nanometer cordyceps militaris peptide. Specifically, in the oral liquid preparation, the content of the nano cordyceps militaris peptide in each 5mg liposome is 500-1500 ug.
The nano cordyceps militaris peptide liposome, the preparation method thereof and the oral liquid preparation are further described in detail with reference to specific embodiments, and the technical scheme of the invention includes but is not limited to the following embodiments.
Example 1
Mixing 50g of soybean hydrogenated lecithin and 50g of cholesterol, dissolving in a mixed solvent of 150g of n-hexane and 150g of chloroform, pouring into a dry round-bottom flask after full dissolution, carrying out rotary evaporation to volatilize an organic solvent, forming a uniform film on the inner wall of the flask, adding 200 ml of a nano cordyceps militaris peptide solution with the pH value of 6.8, hydrating with 83.3 g of water at 50 ℃ for 40 minutes, carrying out ultrasonic treatment for 5 minutes, centrifuging at 4,000 revolutions per minute, taking supernatant, carrying out freeze drying, and canning the obtained white powder.
The transmission electron microscope photograph of the nano cordyceps militaris peptide liposome microsphere is shown in figure 1, wherein figure 1 is a single-molecule photograph of liposome, and the liposome is of a spherical structure. A transmission electron microscope photograph of the nanometer Cordyceps militaris peptide liposome solution is shown in FIG. 2, wherein FIG. 2 shows the distribution state of liposome in water phase, and the particle size range of liposome is 21 nm-57 nm.
Example 2
Mixing 500 g of soybean hydrogenated lecithin and 500 g of cholesterol, dissolving in a mixed solvent of 1500 g of normal hexane and 1500 g of chloroform, pouring into a dry round-bottom flask after full dissolution, carrying out rotary evaporation to volatilize an organic solvent, forming a uniform film on the inner wall of the flask, adding 2000 ml of nano cordyceps militaris peptide solution with the pH value of 6.8, hydrating with 800 g of water at 50 ℃ for 40 minutes, carrying out ultrasonic treatment for 4 minutes, centrifuging at 4,000 rpm, taking supernatant, carrying out freeze drying, and canning the obtained white powder.
The particle size of the liposome is 21-57 nm.
Example 3
Dissolving 50g of soybean hydrogenated lecithin and cholesterol (the ratio is 6: 7) in 150g of mixed solvent of normal hexane and chloroform with the volume ratio of 1: 1, pouring the mixture into a dry round-bottom flask after the soybean hydrogenated lecithin and cholesterol are fully dissolved, removing the organic solvent by rotary evaporation to form a uniform film on the inner wall of the flask, adding a nano cordyceps militaris peptide solution with the pH value of 6.8, hydrating at 50 ℃ for 40min, carrying out ultrasonic treatment for 4.5mim, centrifuging at 4000rpm for 10min, taking the supernatant, carrying out freeze drying, and canning the obtained white powder. The particle size of the liposome is 21-57 nm.
Example 4
(1) Effect of the ratio of the amount of Soybean hydrogenated lecithin to Cholesterol substance on the encapsulation efficiency
The results of the effect of the ratio of the amount of hydrogenated soybean lecithin to the amount of cholesterol substance on the encapsulation efficiency are shown in FIG. 3.
The present experiment examined the encapsulation efficiency at the ratios of the amounts of soybean hydrogenated lecithin and cholesterol material (A) of 6: 3, 6: 5, 6: 7, 6: 9, 6: 11, respectively, and the encapsulation efficiency of liposomes increased and then decreased within the range of the experiment, probably because the film formation was easier at the ratio of 6: 7. Therefore, three levels of 6: 5, 6: 7 and 6: 9 were selected as the levels of the ratio of the amounts of the hydrogenated soybean lecithin and the cholesterol substance in the orthogonal test.
(2) Effect of hydration temperature on encapsulation efficiency
This test examines the encapsulation efficiency at hydration temperatures of 30 ℃, 40 ℃, 50 ℃, 60 ℃, 70 ℃ and 80 ℃. The effect of hydration temperature on encapsulation efficiency is shown in fig. 4, in the test temperature range, the encapsulation efficiency of the liposome is slowly increased and then gradually decreased along with the increase of hydration temperature, because the phase transition temperature of the hydrogenated soybean lecithin is not reached when the temperature is lower, and the fluidity of the membrane is increased and the encapsulation efficiency is reduced when the temperature is too high. Therefore, three levels of 40 ℃, 50 ℃ and 60 ℃ were selected as the temperature levels in the orthogonal test.
(3) Effect of sonication time on encapsulation efficiency
The results of the effect of sonication time on encapsulation efficiency are shown in fig. 5.
The experiment takes the encapsulation efficiency as an index, the encapsulation efficiency when the ultrasonic treatment time is 1.5min, 3min, 4.5min, 6min and 7.5min is considered, the encapsulation efficiency is gradually improved along with the increase of the ultrasonic time in the range of 1.5min to 4.5min and then is rapidly reduced, because the longer ultrasonic time can cause the liposome to be emulsified too finely, so that the encapsulation efficiency is reduced. Therefore, three levels of 3min, 4.5min and 6min were selected as the levels of the ultrasonic time in the orthogonal test.
(4) Influence of pH of Cordyceps militaris peptide solution on encapsulation efficiency
The experiment examines the encapsulation efficiency when the pH of the cordyceps militaris peptide solution is 5.2, 6.0, 6.8, 7.6 and 8.4, the encapsulation efficiency is highest when the pH is 6, and then gradually decreases, and the influence result of the pH of the cordyceps militaris peptide solution on the encapsulation efficiency is shown in fig. 6. Three levels of pH 6.0, 6.8, 7.6 were therefore selected as the levels of pH in the orthogonal experiment.
Results of orthogonal experiments
The influence of each factor on the preparation of the liposome is examined according to the four-factor three-level orthogonal experimental design and by taking the entrapment rate as an index. The results are shown in Table 1. The results of the orthogonal test show that: the sequence of the influence of each factor is C > D > A > B, the optimal preparation conditions are that the quantity ratio (A) of the soybean hydrogenated lecithin to the cholesterol substance is 6: 7, the hydration temperature (B) is 50 ℃, the ultrasonic treatment time (C) is 4.5min, the pH value (D) of the cordyceps militaris peptide solution is 6.8, and the encapsulation rate is 48.14 percent.
TABLE 1 analysis of orthogonal test results
Note: EN is the encapsulation efficiency of the liposome; k1, K2 and K3 are the average encapsulation efficiency of 3 tests with different levels of each factor respectively; r is the range; A. b, C, D
Example 5
A Tyr standard curve is prepared by a Folin-phenol method, the enzyme activity of the trypsin is measured to be 362.52u/mg, the optimum pH of the enzyme is 8.0, and the optimum temperature is 37 ℃. The trypsin tolerance result of the liposome is shown in figure 7, and compared with the liposome, the natural cordyceps militaris peptide effectively enhances the enzyme digestion tolerance of the trypsin. The reason is that the liposome membrane covers the surface of the peptide molecules of the cordyceps militaris, so that the trypsin is prevented from approaching and protecting the enzyme cutting site of the trypsin.
Example 6
By using NBT photo-reduction method, under different test temperature conditions, the activities of the natural Cordyceps militaris peptide and the liposome are increased and then decreased, compared with the natural Cordyceps militaris peptide, the antioxidant activity of the liposome SOD is lower at lower temperature, while the antioxidant activity of the liposome is slightly higher at higher temperature, and the reaction result of the liposome at different temperatures is shown in FIG. 8. The overall change is relatively gentle, and the result shows that the optimal temperature is 40 ℃.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Claims (8)
1. The nano cordyceps militaris peptide liposome comprises nano cordyceps militaris peptide and a liposome membrane layer, wherein the nano cordyceps militaris peptide is wrapped in the liposome membrane layer, and the nano cordyceps militaris peptide liposome is characterized in that the material for feeding the liposome membrane layer comprises the following components in parts by weight: 6-7 parts of soybean hydrogenated lecithin, 6-9 parts of cholesterol, 17-18 parts of normal hexane, 18-19 parts of chloroform and 9-10 parts of water; the raw materials of the liposome membrane layer comprise soybean hydrogenated lecithin and cholesterol;
the preparation method of the nano cordyceps militaris peptide liposome comprises the following steps:
1) mixing the hydrogenated soybean lecithin with cholesterol to obtain a solid mixture;
2) mixing normal hexane and chloroform to obtain a mixed solvent;
3) mixing and dissolving the solid mixture obtained in the step 1) and the mixed solvent obtained in the step 2) to obtain a mixed solution;
4) removing the organic solvent in the mixed solution in the step 3) to obtain a film;
5) mixing the film obtained in the step 4) with the nano cordyceps militaris peptide, hydrating for 35-45 min at 45-50 ℃, and carrying out ultrasonic cell crushing treatment for 4.5-5 min to obtain hydrated liposome;
6) freezing and centrifuging the hydrated liposome obtained in the step 5), and freezing and drying the supernatant to obtain the nano cordyceps militaris peptide liposome; the conditions of freeze drying are as follows: the sublimation temperature is 10-15 ℃, and the time is 4-6 hr; the step 1) and the step 2) are not limited in time sequence;
the preparation method of the nano cordyceps militaris peptide solution comprises the following steps: ball-milling the fruiting body of Cordyceps militaris for 30min to obtain nanometer Cordyceps militaris powder; dissolving the nano cordyceps militaris powder in water, fermenting for 24-36 hours at the pH value of 6.5-8.0 and the temperature of 30-40 ℃, centrifuging for 10-15 min, and performing 5000 dalton molecular weight interception ultrafiltration on the obtained supernatant to obtain a filtrate, namely a nano cordyceps militaris peptide solution;
the content of the nano cordyceps militaris peptide in each 5mg of liposome is 500-1500 mu g.
2. The preparation method of the nano cordyceps militaris peptide liposome as claimed in claim 1, comprising the following steps:
1) mixing the hydrogenated soybean lecithin with cholesterol to obtain a solid mixture;
2) mixing normal hexane and chloroform to obtain a mixed solvent;
3) mixing and dissolving the solid mixture obtained in the step 1) and the mixed solvent obtained in the step 2) to obtain a mixed solution;
4) removing the organic solvent in the mixed solution in the step 3) to obtain a film;
5) mixing the film obtained in the step 4) with the nano cordyceps militaris peptide, hydrating for 35-45 min at 45-50 ℃, and carrying out ultrasonic cell crushing treatment for 4.5-5 min to obtain hydrated liposome;
6) freezing and centrifuging the hydrated liposome obtained in the step 5), and freezing and drying the supernatant to obtain the nano cordyceps militaris peptide liposome; the step 1) and the step 2) are not limited in chronological order.
3. The preparation method of claim 2, wherein the molecular weight of the nano cordyceps militaris peptide of step 5) is not more than 5000 daltons; the nano cordyceps militaris peptide is mixed with the film in a form of nano cordyceps militaris peptide solution, and the pH value of the nano cordyceps militaris peptide solution is 6.5-8.0.
4. The preparation method of claim 2, wherein the ratio of the mass of the nano cordyceps militaris powder to the volume of water is 1-3 g: 30-40 mL.
5. The preparation method according to claim 2, wherein the organic solvent in step 4) is removed by: rotary evaporating at 65-70 deg.c for 20-30 min.
6. The preparation method of claim 2, wherein the added mass of the nano cordyceps militaris peptide in the step 5) is 0.5 times of the mass of the mixed solution in the step 3).
7. The method according to claim 2, wherein the conditions of the step 6) of the refrigerated centrifugation are 0 to 4 ℃ and 3000 to 4000 rpm.
8. An oral liquid preparation containing the nano cordyceps militaris peptide liposome of claim 1 or the nano cordyceps militaris peptide liposome prepared by the preparation method of any one of claims 2 to 7.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1814294A (en) * | 2005-11-29 | 2006-08-09 | 沈阳农业大学 | Corn SOD liposome and its preparing method |
| CN102793177A (en) * | 2012-09-07 | 2012-11-28 | 广州安德生物科技有限公司 | Biological adhesion type cordyceps militaris liposome and preparation method thereof |
| CN103689554A (en) * | 2013-12-25 | 2014-04-02 | 广州安德健康科技有限公司 | Preparation method and application of cordyceps militaris liposome |
| CN104789621A (en) * | 2015-03-18 | 2015-07-22 | 吉林省蚕业科学研究院 | Method for preparing antioxidative peptide through fermentation of cordyceps militaris nanoparticles |
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1814294A (en) * | 2005-11-29 | 2006-08-09 | 沈阳农业大学 | Corn SOD liposome and its preparing method |
| CN102793177A (en) * | 2012-09-07 | 2012-11-28 | 广州安德生物科技有限公司 | Biological adhesion type cordyceps militaris liposome and preparation method thereof |
| CN103689554A (en) * | 2013-12-25 | 2014-04-02 | 广州安德健康科技有限公司 | Preparation method and application of cordyceps militaris liposome |
| CN104789621A (en) * | 2015-03-18 | 2015-07-22 | 吉林省蚕业科学研究院 | Method for preparing antioxidative peptide through fermentation of cordyceps militaris nanoparticles |
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Inventor after: Yang Shufang Inventor after: Wang Ru Inventor after: Xue Qiang Inventor after: Li Sen Inventor after: Jin Xiangdong Inventor after: Zhang Haidong Inventor after: Zhu Xingyou Inventor after: Han Jianhua Inventor after: Sun Jihong Inventor after: Qian Kun Inventor after: Zhang Chunyang Inventor after: Pu Yulan Inventor before: Yang Shufang Inventor before: Wang Ru Inventor before: Xue Qiang Inventor before: Li Sen Inventor before: Zhang Haidong Inventor before: Zhu Xingyou Inventor before: Han Jianhua Inventor before: Sun Jihong Inventor before: Qian Kun Inventor before: Zhang Chunyang Inventor before: Pu Yulan |