CN113769068B - Tanshinone and protein polypeptide composite nanoparticle as well as preparation method and application thereof - Google Patents

Tanshinone and protein polypeptide composite nanoparticle as well as preparation method and application thereof Download PDF

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CN113769068B
CN113769068B CN202111111136.6A CN202111111136A CN113769068B CN 113769068 B CN113769068 B CN 113769068B CN 202111111136 A CN202111111136 A CN 202111111136A CN 113769068 B CN113769068 B CN 113769068B
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tanshinone
nacas
preparation
temperature
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CN113769068A (en
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梁浩
张家奇
王斌
安志敏
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Qinhuangdao Circum Bohai Sea Bio Industry Research Institute Beijing University Of Chemical Technology
Beijing University of Chemical Technology
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Qinhuangdao Circum Bohai Sea Bio Industry Research Institute Beijing University Of Chemical Technology
Beijing University of Chemical Technology
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    • A61K9/51—Nanocapsules; Nanoparticles
    • A61K9/5192—Processes
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    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/02—Stomatological preparations, e.g. drugs for caries, aphtae, periodontitis
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    • A61P31/04—Antibacterial agents
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    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q11/00—Preparations for care of the teeth, of the oral cavity or of dentures; Dentifrices, e.g. toothpastes; Mouth rinses
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
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    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y40/00—Manufacture or treatment of nanostructures
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y5/00—Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery
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    • C12P21/00—Preparation of peptides or proteins
    • C12P21/06—Preparation of peptides or proteins produced by the hydrolysis of a peptide bond, e.g. hydrolysate products
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    • A61K2800/40—Chemical, physico-chemical or functional or structural properties of particular ingredients
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    • A61K2800/805—Corresponding aspects not provided for by any of codes A61K2800/81 - A61K2800/95

Abstract

The invention discloses tanshinone and protein polypeptide composite nanoparticles, and a preparation method and application thereof. The tanshinone lipid-soluble component organic solution is obtained by reacting with natural NaCas solution or enzymolysis NaCas solution at normal temperature, purifying and treating at low temperature, wherein the tanshinone lipid-soluble component is any one of tanshinone IIA, tanshinone I and cryptotanshinone, the enzymolysis NaCas is the NaCas after trypsin enzymolysis, and the normal temperature is 20-35 ℃. The invention can improve the stability and water solubility of tanshinone liposoluble ingredient, and can also provide mineral ions required by teeth remineralization and human body, and enhance the anti-mutans streptococcus activity and antioxidant activity of tanshinone liposoluble ingredient.

Description

Tanshinone and protein polypeptide composite nanoparticle as well as preparation method and application thereof
Technical Field
The invention belongs to the field of pharmaceutical pharmacy research, and particularly relates to a method for preparing water-soluble nanoparticles from tanshinone fat-soluble components and protein polypeptides and application thereof, in particular to a tanshinone and protein polypeptide composite nanoparticle and a preparation method and application thereof.
Background
Caries (tooth decay) is one of the most frequently occurring diseases of humans, and is classified by the World Health Organization (WHO) as one of three major diseases that endanger human health. A large amount of research data at home and abroad indicate that dental caries is a bacterial infectious disease caused by streptococcus mutans and streptococcus marginalis, and streptococcus mutans has been recognized by scholars at home and abroad as the most important and main causative bacteria of dental caries.
Fluoride is currently the most commonly used formulation for preventing caries, but with fluoride use, more and more problems are highlighted. Such as stability of fluorine concentration in fluorine-containing toothpaste, resistance to bacteria fluorine caused by long-term use of fluorine-containing products, mutation of bacterial genome, etc., which cause deterioration of anticaries effect of fluoride; improper use of fluoride can also cause acute and chronic poisoning of organisms, etc., and brings great threat to the health of users, which greatly hinders the popularization of fluoride in the field of caries prevention. The same problem exists in the use of antibiotics to combat caries. The disadvantages of fluoride and antibiotics lead to urgent demands in the market for products for treating and preventing caries, which have good stability and strong killing ability to cariogenic bacteria and are almost harmless to human bodies, as substitutes for fluorine-containing products and antibiotics.
In recent years, research has found that some traditional Chinese herbal medicines (such as red sage root) have the capability of inhibiting bacterial growth and bacterial adhesion, and can achieve an antibacterial effect similar to that of antibiotics. In China, the red sage root, the honeysuckle, the propolis, the platycodon grandiflorum, the liquorice and the like are always considered to have good antibacterial and anti-inflammatory effects in the research of traditional Chinese medicines, and the compound preparation of the red sage root, the honeysuckle, the propolis, the platycodon grandiflorum, the liquorice and the like has practical application in the aspect of oral anti-inflammatory and antibacterial. At present, the active ingredients of the red sage root in the compound reagent in the market are mainly crude tanshinone, and no report of applying the fat-soluble ingredients in the refined crude tanshinone as the main active ingredients to the treatment of dental caries is known at present.
The extract of red sage root has proved to have curative effect in terms of in vitro antibiosis and anti-inflammation. However, the solubility of the extract of Salvia Miltiorrhiza in water is extremely poor, only 10 -7 ～10 -5 mol/L; the red sage root extract has very poor stability and can be decomposed and deactivated under external conditions. This makes the traditional radix Salviae Miltiorrhizae preparation difficult to be absorbed by human body, and has the problem of low bioavailability. Therefore, the search for a preparation method for improving the water-soluble compound of the red sage root extract and the improvement of the water solubility and stability of the red sage root extract are a promising research.
Disclosure of Invention
The invention aims to provide the tanshinone and protein polypeptide composite nanoparticle which can load the tanshinone liposoluble component, improve the stability and water solubility of the tanshinone liposoluble component, provide mineral ions required by teeth remineralization and human body, and enhance the anti-mutans streptococcus activity and the antioxidant activity of the tanshinone liposoluble component. In order to achieve the object, the invention has the following scheme:
the tanshinone and protein polypeptide composite nanoparticle is obtained by reacting a tanshinone fat-soluble component organic solution with NaCas solution or enzymatic hydrolysis NaCas solution at normal temperature, purifying and treating at low temperature, wherein the tanshinone fat-soluble component is any one of tanshinone IIA, tanshinone I and cryptotanshinone, the enzymatic hydrolysis NaCas is NaCas after trypsin enzymolysis, and the normal temperature is 20-35 ℃.
By synthesizing the nano particles, the stability and water solubility of the tanshinone fat-soluble component are improved, and meanwhile, the tanshinone fat-soluble component is easier and more stable to be absorbed and utilized by organisms; naCas can stabilize Ca in the oral cavity 2+ Providing mineral ions required for tooth remineralization. After the enzymolysis protein is combined with tanshinone liposoluble component, 1+1 can be obtained>2 effect. The synthesized nano particles have good stability and water solubility, and the antibacterial performance and the antioxidant capacity are improved.
In a further optimized scheme, the solvent of the tanshinone fat-soluble component organic solution is ethanol, and the NaCas solution or the solvent for enzymolysis of the NaCas solution is water or an alkaline buffer solution. In the normal temperature reaction, the mass ratio of the tanshinone fat-soluble component to the enzymatic hydrolysis NaCas is 1:10 to 1:50. The trypsin is porcine trypsin, and the enzyme activity is 25700U/mg.
If the enzymatic hydrolysis NaCas solution is adopted, the specific preparation method can be as follows:
1) Preparation of NaCas solution: dissolving NaCas in a medium alkaline PBS buffer solution, and placing the solution on a magnetic stirrer to be stirred until the solution is completely dissolved, so as to prepare NaCas solution with the concentration of 10.0-500.0 mg/mL; the pH value of the medium alkaline PBS buffer solution is 6.0-11.0;
2) Preparation of enzymatic hydrolysis NaCas crude solution: adding trypsin into the NaCas solution in the step 1), stirring at 38-80 ℃ by adopting a magnetic stirrer at the rotating speed of 300-1000 rpm, and carrying out enzymolysis for 4-24 h to prepare an enzymolysis NaCas crude solution; the NaCas solution obtained by enzymolysis is different from the CPP solution sold in the market, but the nanoparticle embedding effect and the nanoparticle embedding effect are better than those of the CPP sold in the market;
3) Purification of enzymatic hydrolysis of NaCas solution: placing the enzymatic hydrolysis NaCas crude solution in the step 2) in a boiling water bath, heating for 10min, and inactivating trypsin; centrifugally heating for 10min in a low-temperature refrigerated centrifuge (1-30 ℃) at 4000-7000 rpm, taking supernatant, and discarding precipitate to obtain an enzymolysis NaCas pure solution;
4) Preparation of tanshinone fat-soluble component organic solution: dissolving tanshinone fat-soluble component in absolute ethyl alcohol to obtain tanshinone fat-soluble component organic solution with the concentration of 0.1-1.0 mg/mL;
5) Preparation of enzymatic hydrolysis NaCas@tanshinone crude solution: dropwise adding the tanshinone fat-soluble component organic solution in the step 4) into the enzymolysis NaCas pure solution in the step 3), stirring by a magnetic stirrer while dropwise adding, setting the stirring speed to be 200-1200 rpm, setting the temperature to be 20-35 ℃ after dropwise adding, and setting the speed to be 200-1200 rpm, and magnetically stirring to enable the solution to self-assemble for 2-24 h to obtain the enzymolysis NaCas@tanshinone crude solution;
6) Purifying an enzymatic hydrolysis NaCas@tanshinone solution: placing the enzymatic hydrolysis NaCas@tanshinone crude solution prepared in the step 5) on a rotary evaporator, performing rotary evaporation at the temperature of 30-80 ℃ and the rotating speed of 10-100 rpm, and removing the organic reagent in the solution; then, carrying out freeze centrifugation, centrifuging the solution at the temperature of 1-30 ℃ and under the condition of 2000-6000 g, and taking a supernatant solution to obtain an enzymolysis NaCas@tanshinone pure solution;
7) Obtaining nanoparticles: and (3) performing low-temperature freezing treatment on the pure solution of the enzymatic hydrolysis NaCas@tanshinone in the step (6), wherein the temperature is in the range of 0-80 ℃, obtaining enzymatic hydrolysis NaCas@tanshinone nanoparticles, and then performing freeze drying on the enzymatic hydrolysis NaCas@tanshinone nanoparticles by using a freeze dryer until all solid ice cubes are converted into orange red powder, namely a finished product of the tanshinone and protein polypeptide composite nanoparticles (the crushing shape is a sheet connected together, and the crushing is performed to obtain a powdery product).
If the natural NaCas solution is adopted, the following preparation method can be adopted:
1) Preparation of NaCas solution: dissolving NaCas in a medium alkaline PBS buffer solution, stirring and dissolving to prepare NaCas solution with the concentration of 10.0-500.0 mg/mL; the pH value of the medium alkaline PBS buffer solution is 6.0-11.0;
2) Preparation of tanshinone fat-soluble component organic solution: dissolving tanshinone fat-soluble component in absolute ethyl alcohol to obtain tanshinone fat-soluble component organic solution with the concentration of 0.1-1.0 mg/mL;
3) Preparation of NaCas@tanshinone crude solution: dropwise adding the tanshinone fat-soluble component organic solution in the step 2) into the NaCas solution in the step 1), stirring by a magnetic stirrer while dropwise adding, wherein the stirring speed is 200-1200 rpm, setting the temperature to 20-35 ℃ after dropwise adding, and the speed is 200-1200 rpm, and magnetically stirring to allow the solution to self-assemble for 2-24 h to obtain a NaCas@tanshinone crude solution;
4) Purification of nacas@tanshinone solution: placing the NaCas@tanshinone solution prepared in the step 3) on a rotary evaporator, and performing rotary evaporation at the temperature of 30-80 ℃ and the rotating speed of 10-100 rpm to remove the organic reagent in the solution; centrifuging the solution at the temperature of 1-30 ℃ and under the condition of 2000-6000 g, and taking a supernatant solution to obtain a NaCas@tanshinone pure solution;
5) Obtaining nanoparticles: and (3) performing low-temperature freezing treatment on the NaCas@tanshinone pure solution in the step (4) at a temperature ranging from 0 ℃ to-80 ℃ to obtain NaCas@tanshinone nanoparticles, and then performing freeze drying on the NaCas@tanshinone nanoparticles by using a freeze dryer until all solid ice cubes are converted into powder, thus obtaining the finished product of the tanshinone and protein polypeptide composite nanoparticles.
The protein carriers selected in the research process are natural NaCas and enzymatic NaCas respectively, and the tanshinone fat-soluble components are three, namely tanshinone I, tanshinone IIA and cryptotanshinone respectively. Among them, the best effect of resisting the growth and embedding of mutans streptococcus is enzymolysis NaCas@tanshinone IIA nano particles (MIC=25 mug/mL, embedding rate is more than 99%), and tables 1-3 below are experimental results of various combinations in the study.
TABLE 1
TABLE 2
TABLE 3 Table 3
The invention has the action principle that:
casein is the main protein in mammalian emulsions, and is not only capable of providing calcium ions and essential amino acids for the human body, but also has unique properties in terms of the delivery system of pharmaceutical compounds. Sodium caseinate (NaCas) is a sodium salt of casein, which is negatively charged and is readily attracted by positively charged apatite crystal planes (tooth surfaces); contains Ser (P) -Ser (P) -Ser (P) -Glu-Glu sequence carrying phosphate ion PO 4- Has remarkable effect of stabilizing calcium ions Ca in metastable solution 2+ The capability, easy to form stable CaP, provides sufficient Ca, P and other mineral ions for tooth surface remineralization; naCas is rich in hydrophobic amino acids such as proline, tryptophan and the like, and can encapsulate lipophilic components; meanwhile, the modified amino acid has hydrophilic amino acids such as tyrosine and the like, is easy to dissolve in water, and becomes a natural amphiphilic block copolymer due to the characteristic, and can be used for encapsulating fat-soluble components and improving the water solubility of the fat-soluble components.
The enzymatic hydrolysis process can modify the structure of the protein, thereby improving the functional properties of the protein, and enzymatic hydrolysis of the NaCas can enhance the biological activity of the NaCas, such as antimicrobial properties and antioxidant capacity. When the hydrophobic component is doped into the hydrophobic core of the enzymatic amphiphilic protein nanostructure, the water solubility of the hydrophobic component can be greatly improved. Meanwhile, the NaCas is hydrolyzed to generate bioactive peptide with smaller molecular weight, and the tanshinone liposoluble component is wrapped to form smaller nano particles. By utilizing the electrostatic self-assembly effect, the nanoparticle for enzymolysis of NaCas and tanshinone liposoluble components can be prepared, and the nanoparticle can improve the stability and water solubility of the tanshinone liposoluble extract.
The nanoparticles reduce the size of the tanshinone liposoluble component in the solution, so that the tanshinone liposoluble component better permeates into the biomembrane on the surface of the caries cavity and acts on the mutans streptococcus in the caries cavity, compared with an artificially synthesized high polymer material carrier, the nanoparticles formed by the enzymolysis of NaCas and the tanshinone liposoluble component are smaller in size, have no peculiar smell, and have high nutritional value and good safety. Compared with free tanshinone liposoluble component, the product has greatly raised stability, water solubility, oxidation resistance, antibacterial and anti-biomembrane performance.
The beneficial effects of the invention are as follows:
sodium caseinate (NaCas) is derived from cow milk, contains various necessary amino acids required by human body, and can effectively promote absorption and utilization of divalent mineral ions such as Ca, fe, zn and the like by human body; naCas contains abundant PO 4- Can stabilize Ca in the oral cavity 2+ Providing sufficient Ca for tooth remineralization 2+ And PO (PO) 4- Meanwhile, the calcium phosphate has the characteristics of good biocompatibility, biological activity, slow release rate, alkali release in the hydrolysis process and the like, and is a natural calcium phosphate reservoir.
The product of the invention enhances the water solubility and stability of tanshinone fat-soluble components, so that the tanshinone fat-soluble components are easier to be absorbed by human bodies;
the product enhances the antibacterial capability of the tanshinone fat-soluble component, so that the Minimum Inhibitory Concentration (MIC) and the Minimum Bactericidal Concentration (MBC) of the tanshinone fat-soluble component compound on the streptococcus mutans are obviously enhanced on the basis of the original single free substance;
the product enhances the antioxidant capacity of tanshinone fat-soluble components, and the antioxidant capacity of nanoparticles is obviously enhanced on the basis of original single free substances;
the preparation condition of the product is mild, no high-temperature and high-pressure condition exists, no toxic reagent participates, the loss of the tanshinone fat-soluble component in the preparation process is reduced, and the utilization rate of the tanshinone fat-soluble component is improved.
Compared with single free tanshinone fat-soluble component, the product nanoparticle of the invention improves the high temperature stability, the pH stability, the room temperature storage stability and the illumination stability.
The trypsin hydrolyzes the NaCas, reduces the molecular weight of the NaCas and preserves the peptide chain characteristics of the NaCas. Compared with the artificially synthesized high molecular carrier material, the NaCas carrier has natural source, higher safety, smaller molecular weight and easier absorption by human body. Enzymolysis NaCas@tanshinone IIA nanoparticles in the product provided by the invention have average particle size Z-average: 70+ -5 nm, potential Zeta-potential of-32.6+ -0.5 mV, dispersion index PDI of 0.18. This shows that the enzymatic hydrolysis NaCas@tanshinone IIA nanoparticle has good structural stability and dispersion characteristics.
The nanoparticle has the encapsulation rate of more than 97% under the condition of optimal drug loading ratio, and has high antibacterial activity. Meanwhile, the invention has low production cost, simple process and economy, is very suitable for large-scale industrial production, and the most encapsulation efficiency data are shown in the table 4:
TABLE 4 Table 4
Drawings
FIG. 1 is a picture of an aqueous NaCas@tanshinone IIA nanoparticle solution;
FIG. 2 is a photograph of an aqueous solution of enzymatically hydrolyzed NaCas@tanshinone IIA nanoparticles;
FIG. 3 is a photograph of an aqueous solution of free tanshinone IIA.
Detailed Description
The invention is further described below with reference to the examples and figures. The invention is not limited to the following embodiments. The concentrations and percentages referred to in the following table refer to the liposoluble tanshinone ingredients.
Example 1
A tanshinone and protein polypeptide composite nanoparticle, its preparation method comprises the following steps:
1) Precisely weighing NaCas with a certain mass, stirring to completely dissolve, and dissolving in PBS (PH=6.0) to prepare 100mg/mL aqueous solution;
2) Weighing 0.0032g of trypsin, adding the trypsin into the NaCas aqueous solution, placing the solution under a magnetic stirrer, and stirring the solution at 50 ℃ and 500rpm/min for 4 hours to obtain an enzymolysis NaCas crude solution;
3) Heating the enzymatic hydrolysis NaCas crude solution containing the enzyme in a boiling water bath for 10min; freeze-centrifuging at 5000rpm and 4 ℃ for 10min, discarding the inactivated enzyme precipitate, and taking the supernatant to obtain an enzymatic hydrolysis NaCas pure solution; taking 1mL of enzymolysis NaCas pure solution, adding coomassie brilliant blue solution in a ratio of 1:4, after fully mixing, measuring the absorbance of the mixed solution by an ultraviolet spectrophotometer at the wavelength of 595nm, and comparing the absorbance with a standard curve of the enzymolysis NaCas solution to obtain the enzymolysis NaCas pure solution with the content of 0.8mg/mL; 4) Accurately weighing 10mg tanshinone IIA, and dissolving in 100mL of absolute ethyl alcohol;
5) Taking 24mL of tanshinone IIA ethanol solution; adding 30mL of 0.8mg/mL enzymolysis NaCas pure solution according to the mass ratio of 1:10, placing the solution on a magnetic stirrer, and self-assembling the solution for 2 hours at the temperature of 25 ℃ and the rotating speed of 450rpm/min to obtain enzymolysis NaCas@tanshinone IIA nanoparticle crude solution;
6) Performing rotary evaporation treatment on the enzymolysis NaCas@tanshinone IIA nanoparticle crude solution by a rotary evaporator, wherein the treatment temperature is 43 ℃, the rotation speed is 70rpm/min, and removing an organic reagent in the crude solution; centrifuging the enzymolysis NaCas@tanshinone IIA solution subjected to rotary steaming in a refrigerated centrifuge for 10min at the temperature of 4 ℃ and the rotating speed of 2000g, taking a supernatant solution, discarding orange-red precipitate, and separating assembled nanoparticles from unassembled tanshinone IIA to obtain an enzymolysis NaCas@tanshinone IIA pure solution;
7) And (3) putting the pure solution of the enzymatic NaCas@tanshinone IIA into a refrigerator at the temperature of minus 20 ℃, freezing into ice, and then putting into a freeze dryer until all the enzymatic NaCas@tanshinone IIA nanoparticles are converted into powder.
The encapsulation rate of the enzymatic hydrolysis NaCas@tanshinone IIA prepared by the method is 52.65%.
Example 2
A tanshinone and protein polypeptide composite nanoparticle, its preparation method comprises the following steps:
1) Accurately weighing natural NaCas to prepare a 0.8mg/mL solution;
2) Accurately weighing 10mg tanshinone IIA, and dissolving in 100mL of absolute ethyl alcohol;
3) Taking 24mL of tanshinone IIA ethanol solution; adding 30mL of natural NaCas solution with the mass ratio of 1:10 and the concentration of 0.8mg/mL, placing the solution on a magnetic stirrer, and self-assembling the solution for 2 hours at the temperature of 25 ℃ and the rotating speed of 450rpm/min to obtain natural NaCas@tanshinone IIA nanoparticle crude solution;
4) Performing rotary evaporation treatment on the crude solution of the natural NaCas@tanshinone IIA nanoparticles by a rotary evaporator, wherein the treatment temperature is 43 ℃, the rotation speed is 50rpm/min, and removing an organic reagent in the crude solution; centrifuging the natural NaCas@tanshinone IIA solution subjected to rotary steaming in a refrigerated centrifuge for 10min at the temperature of 4 ℃ and the rotating speed of 2000g, taking a supernatant solution, discarding orange-red precipitate, and separating assembled nanoparticles from unassembled tanshinone IIA to obtain a natural NaCas@tanshinone IIA pure solution;
5) And (3) putting the pure solution of the natural NaCas@tanshinone IIA into a refrigerator at the temperature of minus 20 ℃, freezing into ice, and then putting into a freeze dryer until all the natural NaCas@tanshinone IIA nanoparticles are converted into powder.
The encapsulation rate of the natural NaCas@tanshinone IIA prepared by the method is 50.79%.
The experimental results of example 1 and example 2 are shown in table 5:
TABLE 5
Example 3
A tanshinone and protein polypeptide composite nanoparticle, its preparation method comprises the following steps:
1) Precisely weighing NaCas with a certain mass, stirring to completely dissolve, and dissolving in PBS (PH=7.0) to prepare 300mg/mL aqueous solution;
2) Weighing 0.0096g of trypsin, adding the trypsin into the NaCas aqueous solution, placing the solution under a magnetic stirrer, and stirring the solution at 50 ℃ and 500rpm/min for 12 hours to obtain an enzymolysis NaCas crude solution;
3) Heating the enzymatic NaCas crude solution containing the enzyme in a boiling water bath for 10min, cooling and centrifuging at 5000rpm and 4 ℃ for 10min, discarding the inactivated enzyme precipitate, and taking the supernatant to obtain an enzymatic NaCas pure solution; taking 1mL of enzymolysis NaCas pure solution, adding coomassie brilliant blue solution in a ratio of 1:4, after fully mixing, measuring the absorbance of the mixed solution by an ultraviolet spectrophotometer at the wavelength of 595nm, and comparing the absorbance with a standard curve of the enzymolysis NaCas solution to obtain the enzymolysis NaCas pure solution with the content of 1.6mg/mL;
4) Accurately weighing 20mg of tanshinone IIA, dissolving in 100mL of absolute ethanol, and taking 8mL of tanshinone IIA ethanol solution;
5) Adding tanshinone IIA ethanol solution into 30mL of 1.6mg/mL enzymolysis NaCas solution at a mass ratio of 1:30, placing on a magnetic stirrer, and self-assembling at 25 ℃ and a rotating speed of 450rpm/min for 12h to obtain enzymolysis NaCas@tanshinone IIA nanoparticle crude solution;
6) Performing rotary evaporation treatment on the enzymolysis NaCas@tanshinone IIA nanoparticle crude solution by a rotary evaporator, wherein the treatment temperature is 43 ℃, the rotating speed is 100rpm/min, and removing an organic reagent in the crude solution; centrifuging the enzymolysis NaCas@tanshinone IIA solution subjected to rotary steaming in a refrigerated centrifuge for 10min at the temperature of 4 ℃ and the rotating speed of 2000g, taking a supernatant solution, discarding orange-red precipitate, and separating assembled nanoparticles from unassembled tanshinone IIA to obtain an enzymolysis NaCas@tanshinone IIA pure solution;
7) And (3) putting the pure solution of the enzymatic NaCas@tanshinone IIA into a refrigerator at the temperature of minus 20 ℃, freezing into ice, and then putting into a freeze dryer until all the enzymatic NaCas@tanshinone IIA nanoparticles are converted into powder.
The encapsulation rate of the enzymatic hydrolysis NaCas@tanshinone IIA prepared by the method is 99.09%.
Example 4
A tanshinone and protein polypeptide composite nanoparticle, its preparation method comprises the following steps:
1) Accurately weighing natural NaCas to prepare a 1.6mg/mL solution;
2) Accurately weighing 20mg of tanshinone IIA, and dissolving in 100mL of absolute ethyl alcohol;
3) Taking 8mL of tanshinone IIA ethanol solution; adding 30mL of 1.6mg/mL natural NaCas solution in a mass ratio of 1:30, placing on a magnetic stirrer, and self-assembling for 12h at a temperature of 25 ℃ and a rotating speed of 450rpm/min to obtain a natural NaCas@tanshinone IIA nanoparticle crude solution;
4) Performing rotary evaporation treatment on the crude solution of the natural NaCas@tanshinone IIA nanoparticles by a rotary evaporator, wherein the treatment temperature is 43 ℃, the rotation speed is 100rpm/min, and removing an organic reagent in the crude solution; centrifuging the natural NaCas@tanshinone IIA solution subjected to rotary steaming in a refrigerated centrifuge for 10min at the temperature of 4 ℃ and the rotating speed of 2000g, taking a supernatant solution, discarding orange-red precipitate, and separating assembled nanoparticles from unassembled tanshinone IIA to obtain a natural NaCas@tanshinone IIA pure solution;
5) And (3) putting the pure solution of the natural NaCas@tanshinone IIA into a refrigerator at the temperature of minus 20 ℃, freezing into ice, and then putting into a freeze dryer until all the natural NaCas@tanshinone IIA nanoparticles are converted into powder.
The encapsulation rate of the natural NaCas@tanshinone IIA prepared by the method is 99.02%.
The experimental results of example 3 and example 4 are shown in table 6:
TABLE 6
Example 5
A tanshinone and protein polypeptide composite nanoparticle, its preparation method comprises the following steps:
1) Precisely weighing NaCas with a certain mass, stirring to completely dissolve, and dissolving in PBS (PH=11.0) to prepare 500mg/mL aqueous solution;
2) Weighing 0.016g of trypsin, adding the trypsin into the NaCas aqueous solution, placing the solution under a magnetic stirrer, and stirring the solution at 50 ℃ and 500rpm/min for 24 hours to obtain an enzymatic hydrolysis NaCas crude solution;
3) Heating the enzymatic NaCas crude solution containing the enzyme in a boiling water bath for 10min, cooling and centrifuging at 5000rpm and 4 ℃ for 10min, discarding the inactivated enzyme precipitate, and taking the supernatant to obtain an enzymatic NaCas pure solution; taking 1mL of enzymolysis NaCas pure solution, adding coomassie brilliant blue solution in a ratio of 1:4, after fully mixing, measuring the absorbance of the mixed solution by an ultraviolet spectrophotometer at the wavelength of 595nm, and comparing the absorbance with a standard curve of the enzymolysis NaCas solution to obtain the enzymolysis NaCas pure solution with the content of 2.4mg/mL;
4) Accurately weighing 100mg tanshinone IIA, and dissolving in 100mL of absolute ethyl alcohol;
5) Taking 9.6mL of tanshinone IIA ethanol solution, adding 30mL of 2.4mg/mL of enzymolysis NaCas solution in a mass ratio of 1:50, placing on a magnetic stirrer, and self-assembling for 24h at a temperature of 25 ℃ and a rotating speed of 450rpm to obtain enzymolysis NaCas@tanshinone IIA nanoparticle crude solution;
6) Performing rotary evaporation treatment on the enzymolysis NaCas@tanshinone IIA nanoparticle crude solution by a rotary evaporator, wherein the treatment temperature is 43 ℃, the rotating speed is 100rpm/min, and removing an organic reagent in the crude solution; centrifuging the enzymolysis NaCas@tanshinone IIA solution subjected to rotary steaming in a refrigerated centrifuge for 10min at the temperature of 4 ℃ and the rotating speed of 2000g, taking a supernatant solution, discarding orange-red precipitate, and separating assembled nanoparticles from unassembled tanshinone IIA to obtain an enzymolysis NaCas@tanshinone IIA pure solution;
7) And (3) putting the pure solution of the enzymatic NaCas@tanshinone IIA into a refrigerator at the temperature of minus 20 ℃, freezing into ice, and then putting into a freeze dryer until all the enzymatic NaCas@tanshinone IIA nanoparticles are converted into powder.
The encapsulation rate of the enzymatic hydrolysis NaCas@tanshinone IIA prepared by the method is 62.17%.
Example 6
A tanshinone and protein polypeptide composite nanoparticle, its preparation method comprises the following steps:
1) Accurately weighing natural NaCas to prepare a 2.4mg/mL solution;
2) Accurately weighing 100mg tanshinone IIA, and dissolving in 100mL of absolute ethyl alcohol;
3) Taking 9.6mL of tanshinone IIA solution, adding 30mL of natural NaCas solution with the mass ratio of 1:50, placing the solution on a magnetic stirrer, and self-assembling the solution for 24 hours at the temperature of 25 ℃ and the rotating speed of 450rpm/min to obtain natural NaCas@tanshinone IIA nanoparticle crude solution;
4) Performing rotary evaporation treatment on the crude solution of the natural NaCas@tanshinone IIA nanoparticles by a rotary evaporator, wherein the treatment temperature is 43 ℃, the rotation speed is 100rpm/min, and removing an organic reagent in the crude solution; centrifuging the natural NaCas@tanshinone IIA solution subjected to rotary steaming in a refrigerated centrifuge for 10min at the temperature of 4 ℃ and the rotating speed of 2000g, taking a supernatant solution, discarding orange-red precipitate, and separating assembled nanoparticles from unassembled tanshinone IIA to obtain a natural NaCas@tanshinone IIA pure solution;
5) And (3) putting the pure solution of the natural NaCas@tanshinone IIA into a refrigerator at the temperature of minus 20 ℃, freezing into ice, and then putting into a freeze dryer until all the natural NaCas@tanshinone IIA nanoparticles are converted into powder.
The encapsulation rate of the natural NaCas@tanshinone IIA prepared by the method is 58.65%.
The experimental results of example 5 and example 6 are shown in table 7:
TABLE 7
Example 7
A tanshinone and protein polypeptide composite nanoparticle, its preparation method comprises the following steps:
1) Precisely weighing NaCas with a certain mass, stirring to completely dissolve, and dissolving in PBS (PH=7.0) to prepare 300mg/mL aqueous solution;
2) Weighing 0.0096g of trypsin, adding the trypsin into the NaCas aqueous solution, placing the solution under a magnetic stirrer, and stirring the solution at 50 ℃ and 500rpm/min for 12 hours to obtain an enzymolysis NaCas crude solution;
3) Heating the enzymatic NaCas crude solution containing the enzyme in a boiling water bath for 10min, cooling and centrifuging at 5000rpm and 4 ℃ for 10min, discarding the inactivated enzyme precipitate, and taking the supernatant to obtain an enzymatic NaCas pure solution; taking 1mL of enzymolysis NaCas pure solution, adding coomassie brilliant blue solution in a ratio of 1:4, after fully mixing, measuring the absorbance of the mixed solution by an ultraviolet spectrophotometer at the wavelength of 595nm, and comparing the absorbance with a standard curve of the enzymolysis NaCas solution to obtain the enzymolysis NaCas pure solution with the content of 1.6mg/mL;
4) Accurately weighing 20mg of tanshinone I, and dissolving in 100mL of absolute ethyl alcohol;
5) Taking 8mL of tanshinone I solution, adding 30mL of 1.6mg/mL of enzymolysis NaCas solution in a mass ratio of 1:30, placing on a magnetic stirrer, and self-assembling for 12h at a temperature of 25 ℃ and a rotating speed of 450rpm/min to obtain enzymolysis NaCas@tanshinone I nanoparticle crude solution;
6) Performing rotary evaporation treatment on the enzymolysis NaCas@tanshinone I nanoparticle crude solution by a rotary evaporator, wherein the treatment temperature is 43 ℃, the rotation speed is 100rpm/min, and removing an organic reagent in the crude solution; centrifuging the enzymolysis NaCas@tanshinone I solution subjected to rotary steaming in a refrigerated centrifuge for 10min at the temperature of 4 ℃ and the rotating speed of 2000g, taking a supernatant solution, discarding orange-yellow precipitate, and separating assembled nanoparticles from unassembled tanshinone I to obtain an enzymolysis NaCas@tanshinone I pure solution;
7) And (3) putting the pure solution of the enzymatic hydrolysis NaCas@tanshinone I into a refrigerator at the temperature of minus 20 ℃, freezing into ice, and then putting into a freeze dryer until all the enzymatic hydrolysis NaCas@tanshinone I nanoparticles are converted into powder.
The encapsulation rate of the enzymatic hydrolysis NaCas@tanshinone I prepared by the method is 99.05%.
Example 8
A tanshinone and protein polypeptide composite nanoparticle, its preparation method comprises the following steps:
1) Accurately weighing natural NaCas to prepare a 1.6mg/mL solution;
2) Accurately weighing 20mg of tanshinone I, and dissolving in 100mL of absolute ethyl alcohol;
3) Taking 8mL of tanshinone I solution, adding the tanshinone I solution into 30mL of 1.6mg/mL of natural NaCas solution according to the mass ratio of 1:30, placing the solution on a magnetic stirrer, and self-assembling the solution for 12h at the temperature of 25 ℃ and the rotating speed of 450rpm to obtain a crude solution of natural NaCas@tanshinone I nanoparticles;
4) Performing rotary evaporation treatment on the crude NaCas@tanshinone I nanoparticle solution by a rotary evaporator, wherein the treatment temperature is 43 ℃, the rotation speed is 100rpm/min, and removing an organic reagent in the crude solution; centrifuging the natural NaCas@tanshinone I solution subjected to rotary steaming in a refrigerated centrifuge for 10min at the temperature of 4 ℃ and the rotating speed of 2000g, taking a supernatant solution, discarding orange-yellow precipitate, and separating assembled nanoparticles from unassembled tanshinone I to obtain a natural NaCas@tanshinone I pure solution;
5) The pure solution of the natural NaCas@tanshinone I is put into a refrigerator at the temperature of minus 20 ℃, frozen into ice and then put into a freeze dryer until the natural NaCas@tanshinone I nanoparticles are completely converted into powder.
The encapsulation efficiency of the natural NaCas@tanshinone I prepared by the method is 97.01%.
The experimental results of example 7 and example 8 are shown in table 8:
TABLE 8
Example 9
A tanshinone and protein polypeptide composite nanoparticle, its preparation method comprises the following steps:
1) Precisely weighing NaCas with a certain mass, stirring to completely dissolve, dissolving in PBS (PH=7.0) to prepare 300mg/mL aqueous solution,
2) Weighing 0.0096g of trypsin, adding the trypsin into the NaCas aqueous solution, placing the solution under a magnetic stirrer, and stirring the solution at 50 ℃ and 500rpm/min for 12 hours to obtain an enzymolysis NaCas crude solution;
3) Heating the enzymatic NaCas crude solution containing the enzyme in a boiling water bath for 10min, cooling and centrifuging at 5000rpm and 4 ℃ for 10min, discarding the inactivated enzyme precipitate, and taking the supernatant to obtain an enzymatic NaCas pure solution; taking 1mL of enzymolysis NaCas pure solution, adding coomassie brilliant blue solution in a ratio of 1:4, after fully mixing, measuring the absorbance of the mixed solution by an ultraviolet spectrophotometer at the wavelength of 595nm, and comparing the absorbance with a standard curve of the enzymolysis NaCas solution to obtain the enzymolysis NaCas pure solution with the content of 1.6mg/mL;
4) Accurately weighing 20mg of cryptotanshinone and dissolving in 100mL of absolute ethyl alcohol;
5) Taking 8mL of cryptotanshinone solution, adding 30mL of 1.6mg/mL of enzymolysis NaCas solution in a mass ratio of 1:30, placing on a magnetic stirrer, and self-assembling for 12h at a temperature of 25 ℃ and a rotating speed of 450rpm/min to obtain an enzymolysis NaCas@cryptotanshinone nanoparticle crude solution;
6) Performing rotary evaporation treatment on the enzymolysis NaCas@cryptotanshinone nanoparticle crude solution by a rotary evaporator, wherein the treatment temperature is 43 ℃, the rotating speed is 100rpm/min, and removing an organic reagent in the crude solution; centrifuging the enzymolysis NaCas@cryptotanshinone solution subjected to rotary steaming in a refrigerated centrifuge for 10min at the temperature of 4 ℃ and the rotating speed of 2000g, taking a supernatant solution, discarding orange-red precipitate, and separating assembled nanoparticles from unassembled cryptotanshinone to obtain an enzymolysis NaCas@cryptotanshinone pure solution;
7) And (3) putting the pure solution of the enzymatic NaCas@cryptotanshinone into a refrigerator at the temperature of minus 20 ℃, freezing into ice, and then putting into a freeze dryer until the nano particles of the enzymatic NaCas@cryptotanshinone are completely converted into powder.
The encapsulation rate of the enzymatic hydrolysis NaCas@cryptotanshinone prepared by the method is 97.04%.
Example 10
A tanshinone and protein polypeptide composite nanoparticle, its preparation method comprises the following steps:
1) Accurately weighing natural NaCas to prepare a 1.6mg/mL solution;
2) Accurately weighing 20mg of cryptotanshinone and dissolving in 100mL of absolute ethyl alcohol;
3) Taking 8mL of cryptotanshinone IA solution, adding the cryptotanshinone IA solution into 30mL of 1.6mg/mL of natural NaCas solution according to the mass ratio of 1:30, placing the solution on a magnetic stirrer, and self-assembling the solution for 12h at the temperature of 25 ℃ and the rotating speed of 450rpm to obtain natural NaCas@cryptotanshinone nanoparticle crude solution;
4) Performing rotary evaporation treatment on the crude NaCas@cryptotanshinone nanoparticle solution by a rotary evaporator, wherein the treatment temperature is 43 ℃, the rotation speed is 100rpm/min, and removing an organic reagent in the crude solution; centrifuging the natural NaCas@cryptotanshinone solution subjected to rotary steaming in a refrigerated centrifuge for 10min at the temperature of 4 ℃ and the rotating speed of 2000g, taking a supernatant solution, discarding orange-red precipitate, and separating assembled nanoparticles from unassembled cryptotanshinone to obtain a natural NaCas@cryptotanshinone pure solution;
5) And (3) putting the pure natural NaCas@cryptotanshinone solution into a refrigerator at the temperature of minus 20 ℃, freezing into ice, and then putting into a freeze dryer until all the natural NaCas@cryptotanshinone nanoparticles are converted into powder.
The encapsulation rate of the natural NaCas@cryptotanshinone prepared by the method is 96.08%.
The experimental results of example 9 and example 10 are shown in table 9:
TABLE 9
The average encapsulation rate of the NaCas-tanshinone liposoluble ingredient prepared by the invention reaches more than 97%, and the antibacterial activity is high. Meanwhile, the invention has low production cost, simple process and economy, and is very suitable for large-scale industrial production.
The implementation cases of tanshinone I and cryptotanshinone have no excessive statement because of the large variety of the two proteins and the protein-embedded nanoparticles, but do not represent that the preparation of the two proteins and the tanshinone I and cryptotanshinone-related nanoparticles is only carried out under a constant value condition. The foregoing description of the preferred embodiment of the invention is not intended to be limiting, but rather is intended to cover all modifications, equivalents, and alternatives falling within the spirit and principles of the invention.

Claims (4)

1. A preparation method of tanshinone and protein polypeptide composite nanoparticles is characterized by comprising the following steps: the preparation method comprises the following steps of reacting tanshinone fat-soluble component organic solution with enzymatic hydrolysis NaCas solution at normal temperature, purifying and treating at low temperature:
1) Preparation of NaCas solution: dissolving NaCas in a medium alkaline PBS buffer solution, stirring and dissolving to prepare NaCas solution with the concentration of 10.0-500.0 mg/mL; the pH value of the medium alkaline PBS buffer solution is 6.0-11.0;
2) Preparation of enzymatic hydrolysis NaCas crude solution: adding trypsin into the NaCas solution in the step 1), stirring at 50 ℃ by adopting a magnetic stirrer at the rotating speed of 300-1000 rpm, and carrying out enzymolysis for 4-24 hours to prepare an enzymolysis NaCas crude solution;
3) Purification of enzymatic hydrolysis of NaCas solution: placing the enzymatic hydrolysis NaCas crude solution in the step 2) in a boiling water bath for heating, and inactivating trypsin; centrifuging at 4 ℃ and 4000-7000 rpm, taking supernatant, and discarding precipitate to obtain an enzymatic NaCas pure solution;
4) Preparation of tanshinone fat-soluble component organic solution: dissolving tanshinone fat-soluble component in absolute ethyl alcohol to obtain tanshinone fat-soluble component organic solution with the concentration of 0.1-1.0 mg/mL;
the tanshinone liposoluble component is any one of tanshinone IIA, tanshinone I and cryptotanshinone;
5) Preparation of enzymatic hydrolysis NaCas@tanshinone crude solution: dropwise adding the tanshinone fat-soluble component organic solution in the step 4) into the enzymolysis NaCas pure solution in the step 3), stirring by a magnetic stirrer while dropwise adding, setting the stirring speed to be 200-1200 rpm, setting the temperature to be 25 ℃ after dropwise adding, and magnetically stirring at the speed of 200-1200 rpm to allow the solution to self-assemble for 2-24 h to obtain the enzymolysis NaCas@tanshinone crude solution;
the mass ratio of the tanshinone fat-soluble component to the enzymatic hydrolysis NaCas in the step 5) is 1:10 to 1:50;
6) Purifying an enzymatic hydrolysis NaCas@tanshinone solution: placing the enzymatic hydrolysis NaCas@tanshinone crude solution prepared in the step 5) on a rotary evaporator, performing rotary evaporation at the temperature of 43 ℃ and the rotating speed of 10-100 rpm, and removing the organic reagent in the solution; centrifuging the solution at the temperature of 4 ℃ and under the condition of 2000 g-6000 g, and taking a supernatant solution to obtain an enzymatic hydrolysis NaCas@tanshinone pure solution;
7) Obtaining nanoparticles: and (3) performing low-temperature freezing treatment on the pure solution of the enzymatic hydrolysis NaCas@tanshinone in the step (6), wherein the temperature is-20 ℃, obtaining enzymatic hydrolysis NaCas@tanshinone nanoparticles, and then performing freeze drying on the enzymatic hydrolysis NaCas@tanshinone nanoparticles by using a freeze dryer until all solid ice cubes are converted into powder, thus obtaining the finished product of the tanshinone and protein polypeptide composite nanoparticles.
2. A preparation method of tanshinone and protein polypeptide composite nanoparticles is characterized by comprising the following steps: the tanshinone lipid-soluble component organic solution is obtained by reacting with NaCas solution at normal temperature, purifying and treating at low temperature, and comprises the following preparation steps:
1) Preparation of NaCas solution: dissolving NaCas in a medium alkaline PBS buffer solution, stirring and dissolving to prepare NaCas solution with the concentration of 0.8, 1.6 or 2.4mg/mL; the pH value of the medium alkaline PBS buffer solution is 6.0-11.0;
2) Preparation of tanshinone fat-soluble component organic solution: dissolving tanshinone fat-soluble component in absolute ethyl alcohol to obtain tanshinone fat-soluble component organic solution with the concentration of 0.1-1.0 mg/mL;
the tanshinone liposoluble component is any one of tanshinone IIA, tanshinone I and cryptotanshinone;
3) Preparation of NaCas@tanshinone crude solution: dropwise adding the tanshinone fat-soluble component organic solution in the step 2) into the NaCas solution in the step 1), stirring by a magnetic stirrer while dropwise adding, setting the stirring speed to be 200-1200 rpm, setting the temperature to be 25 ℃ after dropwise adding, and magnetically stirring to enable the solution to self-assemble for 2-24 h, so as to obtain a NaCas@tanshinone crude solution;
the mass ratio of tanshinone fat-soluble component to NaCas in step 3) is 1:10 to 1:50;
4) Purification of nacas@tanshinone solution: placing the NaCas@tanshinone solution prepared in the step 3) on a rotary evaporator, and performing rotary evaporation at the temperature of 43 ℃ and the rotating speed of 10 rpm-100 rpm to remove the organic reagent in the solution; centrifuging the solution at the temperature of 4 ℃ and under the condition of 2000 g-6000 g, and taking a supernatant solution to obtain a NaCas@tanshinone pure solution;
5) Obtaining nanoparticles: and (3) performing low-temperature freezing treatment on the NaCas@tanshinone pure solution in the step (4) at the temperature of-20 ℃ to obtain NaCas@tanshinone nanoparticles, and then performing freeze drying on the NaCas@tanshinone nanoparticles by using a freeze dryer until all solid ice cubes are converted into powder to obtain the finished product of the tanshinone and protein polypeptide composite nanoparticles.
3. A tanshinone and protein polypeptide composite nanoparticle prepared by the preparation method of claim 1 or 2.
4. Use of tanshinone and protein polypeptide composite nanoparticle according to claim 3 in preparation of caries-preventing medicine.
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