CN115429752B - Red sage root injection and its preparation method and application - Google Patents

Red sage root injection and its preparation method and application Download PDF

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CN115429752B
CN115429752B CN202211153846.XA CN202211153846A CN115429752B CN 115429752 B CN115429752 B CN 115429752B CN 202211153846 A CN202211153846 A CN 202211153846A CN 115429752 B CN115429752 B CN 115429752B
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liposome
parts
red sage
polydopamine
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CN115429752A (en
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秦雨程
杨雪忠
阙瑞艳
顾洋
沙丽娟
徐卫东
仲华
李志波
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Changshu Leiyunshang Pharmaceutical Co Ltd
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Changshu Leiyunshang Pharmaceutical Co Ltd
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Abstract

The invention provides a red sage root injection, a preparation method and application thereof, belonging to the technical field of medicines. Decocting Saviae Miltiorrhizae radix in water, precipitating with ethanol, removing tannin by acid-base treatment, decolorizing with active carbon, ultrafiltering, purifying, adding into mixed solvent containing egg yolk lecithin and cholesterol, removing solvent, and dispersing solid in buffer solution to obtain liposome suspension; preparing polydopamine nanoparticles, adding liposome suspension under acidic condition, reacting to obtain polydopamine-Saviae Miltiorrhizae radix liposome, adding fibrin targeting peptide, stirring, reacting to obtain targeting peptide-polydopamine-Saviae Miltiorrhizae radix liposome, adding into injectable water, adjusting pH, bottling, and sterilizing to obtain Saviae Miltiorrhizae radix injectable solution. The invention can quickly and accurately directly reach thrombus focus, the salvia miltiorrhiza releases liposome, and the liposome slowly releases active substances of the salvia miltiorrhiza, thereby playing the roles of long-acting thrombolysis, antioxidation, improving vascular elasticity and the like.

Description

Red sage root injection and its preparation method and application
Technical Field
The invention relates to the technical field of medicines, in particular to a red sage root injection and a preparation method and application thereof.
Background
Thrombosis-like cardiovascular and cerebrovascular diseases have become one of the major diseases that seriously affect human health. Abnormal thrombus block in vivo can block blood vessels, so that blood flow speed is reduced or even stopped, blood supply and oxygen supply of an ischemic area are affected, and tissue ischemia necrosis% is caused, so that human life is threatened. Thrombosis mainly includes arterial thrombosis and venous thrombosis due to the different blood flow rates of different blood vessels. Rupture of atherosclerotic plaques causes aggregation and activation of platelets to promote the occurrence of the coagulation cascade and formation of a fibrin network, ultimately leading to arterial thrombosis. Venous embolism mainly occurs in deep veins, and causes of venous thrombosis mainly include "slow blood flow velocity, high blood coagulation status and vascular endothelial dysfunction". The arterial thrombus and venous thrombus mainly comprise 'activated platelet, water insoluble fibrin, red blood cells and the like'.
The root and rhizome of Salvia Miltiorrhiza bge of Labiatae are dried root and rhizome of Salvia Miltiorrhiza bge. Because of its root Pi Dan, it has the effects of nourishing and strengthening, so the name of Salvia Miltiorrhiza is originally carried in Shennong Ben Cao Jing in Han Dynasty and is listed as the superior product, and belongs to the important medicine for activating blood and regulating menstruation. The main components of the red sage root are tanshinone I, tanshinone II, iso-tanshinone I, iso-tanshinone II, cryptotanshinone, tanshinol, salvianolic acid, protocatechuic aldehyde and the like, and the modern pharmacological research shows that the red sage root has very wide pharmacological activity. Clinically, the red sage root injection has obvious curative effect on cardiovascular and cerebrovascular diseases, and has quick response and safe use. The red sage root preparation may be also tablet, pill, ointment, bolus, tincture, etc. and has fast acting and high curative effect.
In the current national quality standard, only protocatechuic aldehyde is used as an index component for quality control, and the content of protocatechuic aldehyde (C7H 6O 3) is not less than 0.2mg per 1ml measured by high performance liquid chromatography. The preparation method of the standard recorded red sage root injection comprises the following steps: taking 1500g of red sage root, adding water and decocting for three times, wherein the first time is 2 hours, the second time and the third time are 1.5 hours respectively, merging decoctions, filtering, and concentrating the filtrate under reduced pressure to 750ml. Precipitating with ethanol twice to obtain ethanol content of 75% and ethanol content of 85% for the first time, refrigerating and standing, filtering, recovering ethanol from filtrate, concentrating to about 250ml, adding injectable water to 400ml, mixing, refrigerating and standing, filtering, adjusting pH to 6.8 with 10% sodium hydroxide solution, boiling for half an hour, filtering, adding injectable water to 1000ml, bottling, and sterilizing.
For historical reasons, the existing red sage root injection also has problems in terms of quality and clinical safety. The quality problems are mainly represented by that the current red sage root injection product is an extract of the comprehensive components of the traditional Chinese medicinal materials prepared by the traditional process, and the existing red sage root injection product has the defects of undefined active ingredients, unstable quality, unstable clinical curative effect, poor safety and controllability, multiple adverse reactions, difficulty in finding reasons and difficulty in adapting to the requirements of modernization and internationalization of the traditional Chinese medicine due to the insufficient knowledge, the imperfect preparation method and the imperfect quality control method.
The unknown non-quantitative components in the current red sage root injection account for a considerable proportion; some macromolecular impurities such as tannins are difficult to remove, and phenomena such as color deepening, turbidity, precipitation and the like can occur after the macromolecular impurities are placed for a period of time, so that the curative effect and the safety of the preparation are affected. Tannins are the main factor affecting the clarity of injection, and are water-soluble polyphenols with relative molecular weight greater than 1000 (generally 3000-5000), which can act with alkaloid, protein and metal ion in medicine to generate precipitate. For example, it forms intermolecular hydrogen bonds with protein molecules, alkaloids and polysaccharides, generating water-insoluble precipitates; therefore, it is necessary to remove tannins contained in the injection. In general, tannins are easy to cause jaundice and liver necrosis when entering the body, hard and painful local intramuscular injection occur, and the tannins are derivatives of polyhydric phenols and are soluble in alcohol and water.
The prior secondary alcohol precipitation process technology has poor tanning matter removal effect, the obtained product is difficult to be qualified, the selectivity is poor, and a large amount of active ingredients are removed simultaneously. The prior art also comprises a heat treatment cold setting method, a hydroalcoholic method, a glue alcohol method, a polyamide method, a glue alcohol resin method and the like (see Wang Ling and the like for details, journal of Hua western medicine, 2003, no.18, p275; wang Jian and the like, journal of basic-layer traditional Chinese medicines, 2001, no.5, p 51); in the hydroalcoholic method, in order to remove tannins with high rate, the pH value is generally adjusted to 9-10, and a large amount of salvianolic acid component is precipitated and removed at the high pH value; the gum alcohol resin method is best considered only in the case of protocatechuic aldehyde, but in fact, due to the structural similarity of tannins and salvianolic acid components, the macroporous resin takes away a large amount of main active ingredients. These methods for removing tannins have poor selectivity, and remove a large amount of salvianolic acid-type active ingredient at the same time as removing tannins; some methods also introduce other impurities, which degrade the quality of the product.
Along with the continuous and deep understanding of the components of the red sage root, the research in recent years shows that the cardiovascular activity of water-soluble salvianolic acid components (generally with the relative molecular weight smaller than 1000) represented by salvianolic acid B and salvianolic acid A in the red sage root is far stronger than that of salvianic acid A and protocatechuic aldehyde, and the water-soluble salvianolic acid components of the red sage root are the main pharmacodynamic substance basis of the red sage root injection and are accepted by academia. The salvianolic acid has wide and remarkable pharmacological activity on cardiovascular system, has strong antioxidant effect, and can protect myocardial injury caused by ischemia and ischemia reperfusion, inhibit platelet aggregation, improve microcirculation, promote vasodilation and improve blood rheology.
The membrane separation technology has been applied to the production of traditional Chinese medicine preparations since the 80 s. Although this technology starts late and has a small application scale, it has received great attention. Among them, ultrafiltration membranes are most widely used. Mainly relates to the following aspects: 1) Extracting effective parts of the traditional Chinese medicine; 2) Production of oral liquid; 3) Preparing an extract preparation; 4) Removing a heat source; 5) Recovering the effective components from the pharmaceutical wastewater. Hao Li et al (Chinese herbal medicine, 1996, no.8, p 471) have used ultrafiltration membranes with a molecular weight cut-off of 2 ten thousand to carry out the process improvement of applying to the salvia miltiorrhiza injection, and the membrane pore size is too large, so that the effect of removing part of tannins is poor under the relative molecular weight of 5000. Meanwhile, the tannin is removed by only using an ultrafiltration method, the ultrafiltration efficiency is low due to higher content of the tannin, and meanwhile, a great amount of active ingredients are lost due to the effects of adsorption and the like.
The main defect of the existing production technology of the salvia miltiorrhiza injection is that the preparation method is not reasonable, so that the loss of the main active ingredient salvianolic acid A, B is very large; the lack of quality control on the key effective component salvianolic acid A leads to unstable clinical curative effect of the product; incomplete removal of tannins and great potential safety hazard in clinical application.
Disclosure of Invention
The invention aims to provide the red sage root injection and the preparation method and application thereof, which can quickly and accurately directly reach thrombus focus, the red sage root releases liposome, and the liposome slowly releases active substances of the red sage root, thereby having the effects of long-acting thrombolysis, antioxidation, vascular elasticity improvement and the like, and the prepared red sage root injection can play the roles of quick, efficient and long-acting thrombolysis, atherosclerosis alleviation, microcirculation improvement, platelet aggregation inhibition, lipid peroxidation resistance and the like, and has wide application prospect.
The technical scheme of the invention is realized as follows:
the invention provides a preparation method of a red sage root injection, which comprises the steps of decocting red sage root in water, precipitating with alcohol, removing tannins by acid-base treatment, decolorizing with active carbon, ultrafiltering and purifying, adding into a mixed solvent containing egg yolk lecithin and cholesterol, removing the solvent, and dispersing the solid in a buffer solution again to obtain liposome suspension; preparing polydopamine nanoparticles, adding liposome suspension under acidic condition, reacting to obtain polydopamine-Saviae Miltiorrhizae radix liposome, adding fibrin targeting peptide, stirring, reacting to obtain targeting peptide-polydopamine-Saviae Miltiorrhizae radix liposome, adding into injectable water, adjusting pH, bottling, and sterilizing to obtain Saviae Miltiorrhizae radix injectable solution.
As a further improvement of the invention, the method comprises the following steps:
s1, water extraction of the red sage root: decocting Saviae Miltiorrhizae radix with water, filtering, mixing filtrates, and concentrating to obtain Saviae Miltiorrhizae radix water extract;
s2, alcohol precipitation: adding ethanol into the water extract of the red sage root prepared in the step S1, performing first precipitation, filtering, continuously adding ethanol into the filtrate, performing second precipitation, filtering, and performing reduced pressure distillation on the filtrate to recover the ethanol to obtain an ethanol precipitation solution;
s3, acid-base treatment: adjusting the pH value of the alcohol precipitation solution obtained in the step S2 to be alkaline by using alkali liquor, heating and boiling for a first time period, cooling, filtering, adjusting the pH value of the filtrate to be acidic by using acid liquor, heating and boiling for a second time period, cooling, and filtering to obtain filtrate;
s4, decoloring: adding active carbon into the filtrate obtained in the step S3, heating, stirring, and filtering to obtain decolorized solution;
s5, ultrafiltration: ultrafiltering the decolorized solution obtained in the step S4 by using an ultrafiltration membrane to obtain ultrafiltrate;
s6, preparing salvia miltiorrhiza liposome: dissolving egg yolk lecithin and cholesterol in a mixed solvent, dripping the ultrafiltrate prepared in the step S5, stirring for reaction after dripping to obtain a liposome solution, removing the solvent under reduced pressure, freeze-drying, adding a phosphate buffer solution, performing ultrasonic dispersion uniformly to obtain a liposome suspension, and preserving at 0-4 ℃;
S7, preparing polydopamine particles: dissolving dopamine hydrochloride in water, adding ethanol and ammonia water, stirring at high speed for reaction, centrifuging, washing precipitate, and freeze-drying to obtain polydopamine nanoparticles;
s8, preparing polydopamine-red sage root liposome: adjusting the pH value of the salvia miltiorrhiza liposome suspension prepared in the step S6 to be acidic, adding the polydopamine nanoparticles prepared in the step S7, stirring for reaction in a dark place, centrifuging, washing the precipitate, and freeze-drying to obtain polydopamine-salvia miltiorrhiza liposome;
s9, preparing targeted peptide-polydopamine-red sage root liposome: dispersing polydopamine-red sage liposome in water, adding fibrin targeting peptide, mixing, stirring to react, and freeze drying to obtain targeting peptide-polydopamine-red sage liposome;
s10, preparing a red sage root injection: adding the targeting peptide-polydopamine-red sage liposome prepared in the step S9 into water for injection, regulating the pH value to 6.7-6.9, stirring and mixing uniformly, encapsulating, and sterilizing to obtain the red sage root injection.
As a further improvement of the invention, in the step S1, the mass ratio of the red sage root to the water is 1:3-5g/mL, the decoction time is 1-2h, the times of decoction are 2-3 times, and the concentration is carried out until the relative density is 1.3-1.5; the ethanol addition amount in the first precipitation in the step S2 is 65-75% of the ethanol content in the system, and the time is 12-15h; the ethanol content in the second precipitation is 75-85% and the time is 3-5h; the preparation method according to claim 2, wherein the alkali liquor in the step S3 is 1-3mol/L NaOH or KOH solution, the pH value is adjusted to 8-10, and the first time period is 1-2h; the acid liquor is HCl or H with the concentration of 1-3mol/L 2 SO 4 The pH value of the solution is regulated to be 5-6.5, and the second time period is 0.5-1h; the mass ratio of the filtrate to the activated carbon in the step S4 is 100:5-12; the temperature of the heating and stirring is 40-50 ℃ and the time is 20-30min.
As a further improvement of the invention, the pore size of the ultrafiltration membrane in the step S5 is 3000-5000D; in the step S6, the mixed solvent is a mixture of dichloromethane and ethanol, and the volume ratio is 1-3:5; the mass ratio of the egg yolk lecithin to the cholesterol to the ultrafiltrate is 3-5:5-7:20-25, the stirring reaction time is 2-3h, and the pH value of the phosphate buffer solution is 7.2-7.5.
As a further improvement of the invention, the mass ratio of the dopamine hydrochloride, the ethanol and the ammonia water in the step S7 is 1-2:20-40:3-7; the rotating speed of the high-speed stirring is 1200-1500r/min, and the reaction time is 15-30min; the pH value in the step S8 is adjusted to 4-5; the mass ratio of the salvia miltiorrhiza liposome suspension to the polydopamine nanoparticles is 10:1-2; the light-shielding stirring time is 5-7h.
As a further improvement of the present invention, the fibrin-targeting peptide in step S9 is selected from at least one of CREKA lipopeptides, GPRPP lipopeptides, lipopeptides added 1-4 amino acids after CREKA, lipopeptides added 1-4 amino acids after GPRPP, and the mass ratio of the polydopamine-red sage liposome to the fibrin-targeting peptide is 10:2-3, wherein the stirring time is 18-22h; the content of the targeting peptide-polydopamine-red sage liposome in the red sage root injection in the step S10 is 3.5-4.5wt%.
As a further improvement of the invention, the fibrin targeting peptide in the step S5 is a mixture of CREKA lipopeptide and GPRPP lipopeptide, and the mass ratio is 3-5:1.
As a further improvement of the invention, the method specifically comprises the following steps:
s1, water extraction of the red sage root: decocting Saviae Miltiorrhizae radix with water at a mass ratio of 1:3-5g/mL for 1-2h for 2-3 times, filtering, mixing filtrates, and concentrating to relative density of 1.3-1.5 to obtain Saviae Miltiorrhizae radix water extract;
s2, alcohol precipitation: adding ethanol into the water extract of radix Salviae Miltiorrhizae obtained in step S1 until the ethanol content is 65-75%, precipitating for 12-15 hr for the first time, filtering, adding ethanol into the filtrate until the ethanol content is 75-85%, precipitating for the second time for 3-5 hr, filtering, and distilling the filtrate under reduced pressure to recover ethanol to obtain ethanol precipitation solution;
s3, acid-base treatment: regulating pH of the ethanol precipitation solution obtained in step S2 to 8-10 with 1-3mol/L NaOH or KOH solution, heating and boiling for 1-2 hr, cooling, filtering, and filtering with 1-3mol/L HCl or H 2 SO 4 The pH value of the solution is regulated to 5-6.5, the solution is heated and boiled for 0.5-1h, cooled and filtered to obtain filtrate;
s4, decoloring: adding 5-12 parts by weight of activated carbon into 100 parts by weight of the filtrate obtained in the step S3, heating to 40-50 ℃, stirring for 20-30min, and filtering to obtain decolorized solution;
S5, ultrafiltration: ultrafiltering the decolorized solution obtained in the step S4 with an ultrafiltration membrane with a pore diameter of 3000-5000D for 30-60min to obtain ultrafiltrate;
s6, preparing salvia miltiorrhiza liposome: dissolving 3-5 parts by weight of egg yolk lecithin and 5-7 parts by weight of cholesterol in 50-70 parts by weight of mixed solvent, wherein the mixed solvent is a mixture of dichloromethane and ethanol, the volume ratio is 1-3:5, dropwise adding 20-25 parts by weight of the ultrafiltrate prepared in the step S5, stirring and reacting for 2-3 hours after the dropwise adding is finished to obtain a liposome solution, removing the solvent under reduced pressure, freeze-drying, adding 100 parts by weight of phosphate buffer with the pH value of 7.2-7.5, uniformly dispersing by ultrasonic waves to obtain a liposome suspension, and preserving at the temperature of 0-4 ℃;
s7, preparing polydopamine particles: dissolving 1-2 parts by weight of dopamine hydrochloride in 20 parts by weight of water, adding 20-40 parts by weight of ethanol and 3-7 parts by weight of 25-28wt% ammonia water, stirring and reacting for 15-30min at 1200-1500r/min, centrifuging, washing precipitate, and freeze-drying to obtain polydopamine nanoparticles;
s8, preparing polydopamine-red sage root liposome: regulating the pH value of the salvia miltiorrhiza liposome suspension prepared in the step S6 to be 4-5, adding 1-2 parts by weight of polydopamine nanoparticles prepared in the step S7 into 10 parts by weight of the acidified salvia miltiorrhiza liposome suspension, stirring and reacting for 5-7 hours in a dark place, centrifuging, washing the precipitate, and freeze-drying to obtain polydopamine-salvia miltiorrhiza liposome;
S9, preparing targeted peptide-polydopamine-red sage root liposome: dispersing 10 parts by weight of polydopamine-salvia miltiorrhiza liposome in water, adding 2-3 parts by weight of fibrin targeting peptide, uniformly mixing, stirring and reacting for 18-22 hours, and freeze-drying to obtain the targeting peptide-polydopamine-salvia miltiorrhiza liposome;
the fibrin targeting peptide is a mixture of CREKA lipopeptide and GPRPP lipopeptide, and the mass ratio is 3-5:1;
s10, preparing a red sage root injection: adding the targeting peptide-polydopamine-red sage liposome prepared in the step S9 into water for injection, regulating the pH value to 6.7-6.9, stirring and mixing uniformly, filling and sealing, and sterilizing to obtain the red sage root injection, wherein the content of the targeting peptide-polydopamine-red sage root liposome in the red sage root injection is 3.5-4.5wt%.
The invention further protects the red sage root injection prepared by the preparation method.
The invention further protects the application of the salvia miltiorrhiza injection in thrombolysis.
The invention has the following beneficial effects:
the polysaccharide component in the red sage root water extract is removed through alcohol precipitation treatment, so that polysaccharide floccules generated in the later injection are avoided, and the quality of the injection is influenced; the invention can fully destroy tannins, proteins and unstable components in the red sage root extracting solution through acid-base treatment, and simultaneously improve the content of active ingredient tanshinol; the invention firstly removes a large amount of macromolecular polysaccharide, protein, tannin and pigment substances after alcohol precipitation, acid-base treatment and decoloration, and the remaining micromolecular active substances can be obviously purified after ultrafiltration, so that the ultrafiltration efficiency is high, and the obtained active compounds mostly are active compounds of the red sage root, and the ultrafiltration loss is reduced.
Fibrin is a specific marker of thrombus. During thrombosis, thrombin and coagulation factors catalyze the production of fibrin from fibrinogen, while fibrin cross-links to each other to form a dense biopolymer network, thereby protecting the blood clot from mechanical stress and proteolytic attack and activating platelet recruitment, which is known to be present in large amounts at the thrombus site. The lipopeptides CREKA and GPRPP are fibrin targeting lipopeptides, the CREKA is linear and only comprises 5 amino acid residues, in addition, the tail end amino acid-cysteine in the CREKA can be biologically coupled through thiol-maleimide reaction, so that the targeting effect is realized, meanwhile, 1-4 amino acids on the rear end chains of the two can be used as connectors, and the targeting effect of the lipopeptides is not influenced.
The fibrin targeting peptide is a mixture of CREKA and GPRPP, is connected with the polydopamine-salvia miltiorrhiza liposome compound, and combines the two, under the synergistic effect, the targeting speed and accuracy are improved, so that the salvia miltiorrhiza liposome can realize efficient thrombus homing, the polydopamine-salvia miltiorrhiza liposome compound can be selectively released in the environment of thrombus, the rapid targeting drug release of the salvia miltiorrhiza extract is realized, and the excellent thrombolysis effect and the good biological safety are shown.
In the salvia miltiorrhiza injection prepared by the invention, active substances of the salvia miltiorrhiza comprise sodium salvianic acid, protocatechuic aldehyde, rosmarinic acid, salvianolic acid B, salvianolic acid A and the like, the active substances of the salvia miltiorrhiza are wrapped in a liposome, hydroxyl on the surface of the liposome and amino on the surface of polydopamine can be electrostatically adsorbed, so that a stable polydopamine-salvia miltiorrhiza liposome compound is formed, and then the amino on the surface of polydopamine in polydopamine-salvia miltiorrhiza liposome can be electrostatically adsorbed with fibrin targeting peptides, such as carboxylic acid on lipopeptid CREKA and GPRPP, so that a stable targeting peptide-polydopamine-salvia miltiorrhiza liposome is formed, the targeting peptide-polydopamine-salvia miltiorrhiza liposome can quickly and accurately directly reach thrombus focus under the action of the targeting peptide, the salvia miltiorrhiza liposome is released, and the active substances of the salvia miltiorrhiza are slowly released, so that the effects of long-acting thrombolysis, antioxidation and the improvement of vascular elasticity are realized, and the prepared salvia miltiorrhiza injection can play the roles of quick, high-efficiency and long-acting thrombolysis, relieving atherosclerosis, improving microcirculation, inhibiting platelet aggregation, resisting lipid peroxidation and the like.
The invention has the advantages that the active substances of the red sage root are embedded by the liposome, so that the effect of better stabilizing the active substances of the red sage root can be achieved, the problems of medicine inactivation, medicine effect reduction and the like caused by long-time storage are avoided, and the invention has obvious practical significance.
Detailed Description
The following description of the technical solutions in the embodiments of the present invention will be clear and complete, and it is obvious that the described embodiments are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
CREKA (Cys-Arg-Glu-Lys-Ala) lipopeptides were purchased from Medium peptide Biochemical Co., ltd; GPRPP (Gly-Pro-Arg-Pro-Pro) lipopeptides were purchased from Kirsrui Biotech. Egg yolk lecithin with purity >90% was purchased from the biological technology limited company of chinese schrader, beijing; cholesterol, purity >95%, was purchased from Shanghai Ala Biochemical technologies Co., ltd.
Example 1
The embodiment provides a preparation method of a red sage root injection, which specifically comprises the following steps:
s1, water extraction of the red sage root: decocting Saviae Miltiorrhizae radix in water for 1h at a mass ratio of Saviae Miltiorrhizae radix to water of 1:3g/mL for 2 times, filtering with 60 mesh sieve, mixing filtrates, and concentrating to relative density of 1.3 to obtain Saviae Miltiorrhizae radix water extract;
s2, alcohol precipitation: adding ethanol into the water extract of the red sage root prepared in the step S1 until the ethanol content in the system is 65%, performing first precipitation for 12 hours, filtering, continuously adding ethanol into the filtrate until the ethanol content in the system is 75%, performing second precipitation for 3 hours, filtering, and performing reduced pressure distillation on the filtrate to recover ethanol to obtain an ethanol precipitation solution;
S3, acid-base treatment: adjusting the pH value of the alcohol precipitation solution obtained in the step S2 to 8 by using a 1mol/L NaOH solution, heating and boiling for 1h, cooling to room temperature, filtering, adjusting the pH value of the filtrate to 6.5 by using a 1mol/L HCl solution, heating and boiling for 0.5h, cooling to room temperature, and filtering to obtain filtrate;
s4, decoloring: adding 5 parts by weight of activated carbon into 100 parts by weight of the filtrate obtained in the step S3, heating to 40 ℃, stirring for 20min, and filtering to obtain decolorized solution;
s5, ultrafiltration: ultrafiltering the decolorized solution obtained in the step S4 with an ultrafiltration membrane with the aperture of 3000D for 30min to obtain ultrafiltrate;
s6, preparing salvia miltiorrhiza liposome: dissolving 3 parts by weight of egg yolk lecithin and 5 parts by weight of cholesterol in 50 parts by weight of a mixed solvent, wherein the mixed solvent is a mixture of dichloromethane and ethanol, the volume ratio is 1:5, dropwise adding 20 parts by weight of the ultrafiltrate prepared in the step S5, stirring for reaction for 2 hours after the dropwise adding is finished to obtain a liposome solution, removing the solvent under reduced pressure, freeze-drying, adding 100 parts by weight of a phosphate buffer solution with the pH value of 7.2, performing 1000W ultrasonic dispersion for 30 minutes to obtain a liposome suspension, and preserving at the temperature of 0 ℃;
s7, preparing polydopamine particles: dissolving 1 part by weight of dopamine hydrochloride in 20 parts by weight of water, adding 20 parts by weight of ethanol and 3 parts by weight of 25wt% ammonia water, stirring for reaction for 15min at 1200r/min, centrifuging for 15min at 5000r/min, washing precipitate with deionized water, and freeze-drying to obtain polydopamine nanoparticles;
S8, preparing polydopamine-red sage root liposome: regulating the pH value of the salvia miltiorrhiza liposome suspension prepared in the step S6 to be 4, adding 1 part by weight of polydopamine nanoparticles prepared in the step S7 into 10 parts by weight of the acidified salvia miltiorrhiza liposome suspension, carrying out light-shielding stirring reaction for 5 hours, centrifuging for 15 minutes at 5000r/min, washing the precipitate by deionized water, and freeze-drying to obtain polydopamine-salvia miltiorrhiza liposome;
s9, preparing targeted peptide-polydopamine-red sage root liposome: dispersing 10 parts by weight of polydopamine-salvia miltiorrhiza liposome in 50 parts by weight of water, adding 2 parts by weight of fibrin targeting peptide, uniformly mixing, stirring for reacting for 18 hours, and freeze-drying to obtain the targeting peptide-polydopamine-salvia miltiorrhiza liposome;
the fibrin targeting peptide is a mixture of CREKA lipopeptide and GPRPP lipopeptide, and the mass ratio is 3:1;
s10, preparing a red sage root injection: adding the targeting peptide-polydopamine-red sage root liposome prepared in the step S9 into water for injection, regulating the pH value to 6.7, stirring, uniformly mixing, filling, sealing and sterilizing to obtain the red sage root injection, wherein the content of the targeting peptide-polydopamine-red sage root liposome in the red sage root injection is 3.5wt%.
Example 2
The embodiment provides a preparation method of a red sage root injection, which specifically comprises the following steps:
S1, water extraction of the red sage root: decocting Saviae Miltiorrhizae radix in water for 2 hr for 3 times, filtering with 60 mesh sieve, mixing filtrates, and concentrating to relative density of 1.5 to obtain Saviae Miltiorrhizae radix water extract;
s2, alcohol precipitation: adding ethanol into the water extract of the red sage root prepared in the step S1 until the ethanol content in the system is 75%, performing first precipitation for 15h, filtering, continuously adding ethanol into the filtrate until the ethanol content in the system is 85%, performing second precipitation for 5h, filtering, and performing reduced pressure distillation on the filtrate to recover ethanol to obtain an ethanol precipitation solution;
s3, acid-base treatment: regulating pH of the ethanol precipitation solution obtained in step S2 to 10 with 3mol/L KOH solution, heating and boiling for 2 hr, cooling to room temperature, filtering, and filtering with 3mol/L H 2 SO 4 The pH value of the solution is regulated to 5, the solution is heated and boiled for 1h, cooled to room temperature and filtered to obtain filtrate;
s4, decoloring: adding 12 parts by weight of active carbon into 100 parts by weight of the filtrate obtained in the step S3, heating to 50 ℃, stirring for 30min, and filtering to obtain decolorized solution;
s5, ultrafiltration: ultrafiltering the decolorized solution obtained in the step S4 with 5000D ultrafiltration membrane for 60min to obtain ultrafiltrate;
s6, preparing salvia miltiorrhiza liposome: dissolving 5 parts by weight of egg yolk lecithin and 7 parts by weight of cholesterol in 70 parts by weight of a mixed solvent, wherein the mixed solvent is a mixture of dichloromethane and ethanol, the volume ratio is 3:5, dropwise adding 25 parts by weight of the ultrafiltrate prepared in the step S5, stirring for reaction for 3 hours after the dropwise adding is finished to obtain a liposome solution, removing the solvent under reduced pressure, freeze-drying, adding 100 parts by weight of a phosphate buffer solution with the pH value of 7.5, performing 1000W ultrasonic dispersion for 30 minutes to obtain a liposome suspension, and preserving at 4 ℃;
S7, preparing polydopamine particles: dissolving 2 parts by weight of dopamine hydrochloride in 20 parts by weight of water, adding 40 parts by weight of ethanol and 7 parts by weight of 28wt% ammonia water, stirring for reaction for 30min at 1500r/min, centrifuging for 15min at 5000r/min, washing precipitate with deionized water, and freeze-drying to obtain polydopamine nanoparticles;
s8, preparing polydopamine-red sage root liposome: regulating the pH value of the salvia miltiorrhiza liposome suspension prepared in the step S6 to be 5, adding 2 parts by weight of polydopamine nanoparticles prepared in the step S7 into 10 parts by weight of the acidified salvia miltiorrhiza liposome suspension, stirring and reacting for 7 hours in a dark place, centrifuging for 15 minutes at 5000r/min, washing the precipitate by deionized water, and freeze-drying to obtain polydopamine-salvia miltiorrhiza liposome;
s9, preparing targeted peptide-polydopamine-red sage root liposome: dispersing 10 parts by weight of polydopamine-salvia miltiorrhiza liposome in 50 parts by weight of water, adding 3 parts by weight of fibrin targeting peptide, uniformly mixing, stirring for reacting for 22 hours, and freeze-drying to obtain the targeting peptide-polydopamine-salvia miltiorrhiza liposome;
the fibrin targeting peptide is a mixture of CREKA lipopeptide and GPRPP lipopeptide, and the mass ratio is 5:1;
s10, preparing a red sage root injection: adding the targeting peptide-polydopamine-red sage root liposome prepared in the step S9 into water for injection, regulating the pH value to 6.9, stirring, uniformly mixing, filling, sealing and sterilizing to obtain the red sage root injection, wherein the content of the targeting peptide-polydopamine-red sage root liposome in the red sage root injection is 4.5wt%.
Example 3
The embodiment provides a preparation method of a red sage root injection, which specifically comprises the following steps:
s1, water extraction of the red sage root: decocting Saviae Miltiorrhizae radix with water at a mass ratio of Saviae Miltiorrhizae radix to water of 1:4g/mL for 1.5h for 3 times, filtering with 60 mesh sieve, mixing filtrates, and concentrating to relative density of 1.4 to obtain Saviae Miltiorrhizae radix water extract;
s2, alcohol precipitation: adding ethanol into the water extract of the red sage root prepared in the step S1 until the ethanol content in the system is 70%, performing first precipitation for 13h, filtering, continuously adding ethanol into the filtrate until the ethanol content in the system is 80%, performing second precipitation for 4h, filtering, and performing reduced pressure distillation on the filtrate to recover ethanol to obtain an ethanol precipitation solution;
s3, acid-base treatment: regulating the pH value of the alcohol precipitation solution obtained in the step S2 to 9 by using 2mol/L NaOH solution, heating and boiling for 1.5h, cooling to room temperature, filtering, regulating the pH value of the filtrate to 6 by using 2mol/L HCl solution, heating and boiling for 1h, cooling to room temperature, and filtering to obtain filtrate;
s4, decoloring: adding 8 parts by weight of activated carbon into 100 parts by weight of the filtrate obtained in the step S3, heating to 45 ℃, stirring for 25min, and filtering to obtain decolorized solution;
s5, ultrafiltration: ultrafiltering the decolorized solution obtained in the step S4 with 4000D ultrafiltration membrane with aperture for 45min to obtain ultrafiltrate;
S6, preparing salvia miltiorrhiza liposome: dissolving 4 parts by weight of egg yolk lecithin and 6 parts by weight of cholesterol in 60 parts by weight of a mixed solvent, wherein the mixed solvent is a mixture of dichloromethane and ethanol, the volume ratio is 2:5, dropwise adding 22 parts by weight of the ultrafiltrate prepared in the step S5, stirring and reacting for 2.5 hours after the dropwise adding is finished to obtain a liposome solution, removing the solvent under reduced pressure, freeze-drying, adding 100 parts by weight of a phosphate buffer solution with the pH value of 7.3, performing 1000W ultrasonic dispersion for 30min to obtain a liposome suspension, and preserving at 2 ℃;
s7, preparing polydopamine particles: dissolving 1.5 parts by weight of dopamine hydrochloride in 20 parts by weight of water, adding 30 parts by weight of ethanol and 5 parts by weight of 27wt% ammonia water, stirring for reaction for 22min at 1350r/min, centrifuging for 15min at 5000r/min, washing precipitate with deionized water, and freeze-drying to obtain polydopamine nanoparticles;
s8, preparing polydopamine-red sage root liposome: regulating the pH value of the salvia miltiorrhiza liposome suspension prepared in the step S6 to be 4.5, adding 1.5 parts by weight of polydopamine nanoparticles prepared in the step S7 into 10 parts by weight of the acidified salvia miltiorrhiza liposome suspension, stirring and reacting for 6 hours in a dark place, centrifuging for 15 minutes at 5000r/min, washing the precipitate with deionized water, and freeze-drying to obtain polydopamine-salvia miltiorrhiza liposome;
S9, preparing targeted peptide-polydopamine-red sage root liposome: dispersing 10 parts by weight of polydopamine-red sage root liposome in 50 parts by weight of water, adding 2.5 parts by weight of fibrin targeting peptide, uniformly mixing, stirring for reacting for 20 hours, and freeze-drying to obtain the targeting peptide-polydopamine-red sage root liposome;
the fibrin targeting peptide is a mixture of CREKA lipopeptide and GPRPP lipopeptide, and the mass ratio is 4:1;
s10, preparing a red sage root injection: adding the targeting peptide-polydopamine-red sage root liposome prepared in the step S9 into water for injection, regulating the pH value to 6.8, stirring, uniformly mixing, filling, sealing and sterilizing to obtain the red sage root injection, wherein the content of the targeting peptide-polydopamine-red sage root liposome in the red sage root injection is 4wt%.
Example 4
In contrast to example 3, the fibrin-targeting peptide was a single CREKA lipopeptide, with no other conditions being altered.
Example 5
In contrast to example 3, the fibrin-targeting peptide was a single GPRPP lipopeptide, with no change in other conditions.
Comparative example 1
In contrast to example 3, only the first precipitation was included in step S2, and the other conditions were not changed.
The method specifically comprises the following steps:
s1, water extraction of the red sage root: decocting Saviae Miltiorrhizae radix with water at a mass ratio of Saviae Miltiorrhizae radix to water of 1:4g/mL for 1.5h for 3 times, filtering with 60 mesh sieve, mixing filtrates, and concentrating to relative density of 1.4 to obtain Saviae Miltiorrhizae radix water extract;
S2, alcohol precipitation: adding ethanol into the water extract of the red sage root prepared in the step S1 until the ethanol content in the system is 70%, precipitating for 13h, filtering, and distilling the filtrate under reduced pressure to recover ethanol to obtain an ethanol precipitation solution;
s3, acid-base treatment: regulating the pH value of the alcohol precipitation solution obtained in the step S2 to 9 by using 2mol/L NaOH solution, heating and boiling for 1.5h, cooling to room temperature, filtering, regulating the pH value of the filtrate to 6 by using 2mol/L HCl solution, heating and boiling for 1h, cooling to room temperature, and filtering to obtain filtrate;
s4, decoloring: adding 8 parts by weight of activated carbon into 100 parts by weight of the filtrate obtained in the step S3, heating to 45 ℃, stirring for 25min, and filtering to obtain decolorized solution;
s5, ultrafiltration: ultrafiltering the decolorized solution obtained in the step S4 with 4000D ultrafiltration membrane with aperture for 45min to obtain ultrafiltrate;
s6, preparing salvia miltiorrhiza liposome: dissolving 4 parts by weight of egg yolk lecithin and 6 parts by weight of cholesterol in 60 parts by weight of a mixed solvent, wherein the mixed solvent is a mixture of dichloromethane and ethanol, the volume ratio is 2:5, dropwise adding 22 parts by weight of the ultrafiltrate prepared in the step S5, stirring and reacting for 2.5 hours after the dropwise adding is finished to obtain a liposome solution, removing the solvent under reduced pressure, freeze-drying, adding 100 parts by weight of a phosphate buffer solution with the pH value of 7.3, performing 1000W ultrasonic dispersion for 30min to obtain a liposome suspension, and preserving at 2 ℃;
S7, preparing polydopamine particles: dissolving 1.5 parts by weight of dopamine hydrochloride in 20 parts by weight of water, adding 30 parts by weight of ethanol and 5 parts by weight of 27wt% ammonia water, stirring for reaction for 22min at 1350r/min, centrifuging for 15min at 5000r/min, washing precipitate with deionized water, and freeze-drying to obtain polydopamine nanoparticles;
s8, preparing polydopamine-red sage root liposome: regulating the pH value of the salvia miltiorrhiza liposome suspension prepared in the step S6 to be 4.5, adding 1.5 parts by weight of polydopamine nanoparticles prepared in the step S7 into 10 parts by weight of the acidified salvia miltiorrhiza liposome suspension, stirring and reacting for 6 hours in a dark place, centrifuging for 15 minutes at 5000r/min, washing the precipitate with deionized water, and freeze-drying to obtain polydopamine-salvia miltiorrhiza liposome;
s9, preparing targeted peptide-polydopamine-red sage root liposome: dispersing 10 parts by weight of polydopamine-red sage root liposome in 50 parts by weight of water, adding 2.5 parts by weight of fibrin targeting peptide, uniformly mixing, stirring for reacting for 20 hours, and freeze-drying to obtain the targeting peptide-polydopamine-red sage root liposome;
the fibrin targeting peptide is a mixture of CREKA lipopeptide and GPRPP lipopeptide, and the mass ratio is 4:1;
s10, preparing a red sage root injection: adding the targeting peptide-polydopamine-red sage root liposome prepared in the step S9 into water for injection, regulating the pH value to 6.8, stirring, uniformly mixing, filling, sealing and sterilizing to obtain the red sage root injection, wherein the content of the targeting peptide-polydopamine-red sage root liposome in the red sage root injection is 4wt%.
Comparative example 2
In contrast to example 3, only the second precipitation was included in step S2, and the other conditions were not changed.
The method specifically comprises the following steps:
s1, water extraction of the red sage root: decocting Saviae Miltiorrhizae radix with water at a mass ratio of Saviae Miltiorrhizae radix to water of 1:4g/mL for 1.5h for 3 times, filtering with 60 mesh sieve, mixing filtrates, and concentrating to relative density of 1.4 to obtain Saviae Miltiorrhizae radix water extract;
s2, alcohol precipitation: adding ethanol into the water extract of the red sage root prepared in the step S1 until the ethanol content in the system is 80%, precipitating for 4 hours, filtering, and distilling the filtrate under reduced pressure to recover ethanol to obtain an ethanol precipitation solution;
s3, acid-base treatment: regulating the pH value of the alcohol precipitation solution obtained in the step S2 to 9 by using 2mol/L NaOH solution, heating and boiling for 1.5h, cooling to room temperature, filtering, regulating the pH value of the filtrate to 6 by using 2mol/L HCl solution, heating and boiling for 1h, cooling to room temperature, and filtering to obtain filtrate;
s4, decoloring: adding 8 parts by weight of activated carbon into 100 parts by weight of the filtrate obtained in the step S3, heating to 45 ℃, stirring for 25min, and filtering to obtain decolorized solution;
s5, ultrafiltration: ultrafiltering the decolorized solution obtained in the step S4 with 4000D ultrafiltration membrane with aperture for 45min to obtain ultrafiltrate;
s6, preparing salvia miltiorrhiza liposome: dissolving 4 parts by weight of egg yolk lecithin and 6 parts by weight of cholesterol in 60 parts by weight of a mixed solvent, wherein the mixed solvent is a mixture of dichloromethane and ethanol, the volume ratio is 2:5, dropwise adding 22 parts by weight of the ultrafiltrate prepared in the step S5, stirring and reacting for 2.5 hours after the dropwise adding is finished to obtain a liposome solution, removing the solvent under reduced pressure, freeze-drying, adding 100 parts by weight of a phosphate buffer solution with the pH value of 7.3, performing 1000W ultrasonic dispersion for 30min to obtain a liposome suspension, and preserving at 2 ℃;
S7, preparing polydopamine particles: dissolving 1.5 parts by weight of dopamine hydrochloride in 20 parts by weight of water, adding 30 parts by weight of ethanol and 5 parts by weight of 27wt% ammonia water, stirring for reaction for 22min at 1350r/min, centrifuging for 15min at 5000r/min, washing precipitate with deionized water, and freeze-drying to obtain polydopamine nanoparticles;
s8, preparing polydopamine-red sage root liposome: regulating the pH value of the salvia miltiorrhiza liposome suspension prepared in the step S6 to be 4.5, adding 1.5 parts by weight of polydopamine nanoparticles prepared in the step S7 into 10 parts by weight of the acidified salvia miltiorrhiza liposome suspension, stirring and reacting for 6 hours in a dark place, centrifuging for 15 minutes at 5000r/min, washing the precipitate with deionized water, and freeze-drying to obtain polydopamine-salvia miltiorrhiza liposome;
s9, preparing targeted peptide-polydopamine-red sage root liposome: dispersing 10 parts by weight of polydopamine-red sage root liposome in 50 parts by weight of water, adding 2.5 parts by weight of fibrin targeting peptide, uniformly mixing, stirring for reacting for 20 hours, and freeze-drying to obtain the targeting peptide-polydopamine-red sage root liposome;
the fibrin targeting peptide is a mixture of CREKA lipopeptide and GPRPP lipopeptide, and the mass ratio is 4:1;
s10, preparing a red sage root injection: adding the targeting peptide-polydopamine-red sage root liposome prepared in the step S9 into water for injection, regulating the pH value to 6.8, stirring, uniformly mixing, filling, sealing and sterilizing to obtain the red sage root injection, wherein the content of the targeting peptide-polydopamine-red sage root liposome in the red sage root injection is 4wt%.
Comparative example 3
In contrast to example 3, the step S2 alcohol precipitation step was not performed, and the other conditions were not changed.
The method specifically comprises the following steps:
s1, water extraction of the red sage root: decocting Saviae Miltiorrhizae radix with water at a mass ratio of Saviae Miltiorrhizae radix to water of 1:4g/mL for 1.5h for 3 times, filtering with 60 mesh sieve, mixing filtrates, and concentrating to relative density of 1.4 to obtain Saviae Miltiorrhizae radix water extract;
s2, acid-base treatment: regulating pH of the water extract of radix Salviae Miltiorrhizae obtained in step S1 to 9 with 2mol/L NaOH solution, boiling for 1.5 hr, cooling to room temperature, filtering, regulating pH of the filtrate to 6 with 2mol/L HCl solution, boiling for 1 hr, cooling to room temperature, and filtering to obtain filtrate;
s3, decoloring: adding 8 parts by weight of activated carbon into 100 parts by weight of the filtrate obtained in the step S2, heating to 45 ℃, stirring for 25min, and filtering to obtain decolorized solution;
s4, ultrafiltration: ultrafiltering the decolorized solution obtained in the step S3 with 4000D ultrafiltration membrane with aperture for 45min to obtain ultrafiltrate;
s5, preparing salvia miltiorrhiza liposome: dissolving 4 parts by weight of egg yolk lecithin and 6 parts by weight of cholesterol in 60 parts by weight of a mixed solvent, wherein the mixed solvent is a mixture of dichloromethane and ethanol, the volume ratio is 2:5, dropwise adding 22 parts by weight of the ultrafiltrate prepared in the step S4, stirring and reacting for 2.5 hours after the dropwise adding is finished to obtain a liposome solution, removing the solvent under reduced pressure, freeze-drying, adding 100 parts by weight of a phosphate buffer solution with the pH value of 7.3, performing 1000W ultrasonic dispersion for 30min to obtain a liposome suspension, and preserving at 2 ℃;
S6, preparing polydopamine particles: dissolving 1.5 parts by weight of dopamine hydrochloride in 20 parts by weight of water, adding 30 parts by weight of ethanol and 5 parts by weight of 27wt% ammonia water, stirring for reaction for 22min at 1350r/min, centrifuging for 15min at 5000r/min, washing precipitate with deionized water, and freeze-drying to obtain polydopamine nanoparticles;
s7, preparing polydopamine-red sage root liposome: regulating the pH value of the salvia miltiorrhiza liposome suspension prepared in the step S5 to be 4.5, adding 1.5 parts by weight of polydopamine nanoparticles prepared in the step S6 into 10 parts by weight of the acidified salvia miltiorrhiza liposome suspension, stirring and reacting for 6 hours in a dark place, centrifuging for 15 minutes at 5000r/min, washing the precipitate with deionized water, and freeze-drying to obtain polydopamine-salvia miltiorrhiza liposome;
s8, preparing targeted peptide-polydopamine-red sage root liposome: dispersing 10 parts by weight of polydopamine-red sage root liposome in 50 parts by weight of water, adding 2.5 parts by weight of fibrin targeting peptide, uniformly mixing, stirring for reacting for 20 hours, and freeze-drying to obtain the targeting peptide-polydopamine-red sage root liposome;
the fibrin targeting peptide is a mixture of CREKA lipopeptide and GPRPP lipopeptide, and the mass ratio is 4:1;
s9, preparing a red sage root injection: adding the targeting peptide-polydopamine-red sage root liposome prepared in the step S8 into water for injection, regulating the pH value to 6.8, stirring, uniformly mixing, filling, sealing and sterilizing to obtain the red sage root injection, wherein the content of the targeting peptide-polydopamine-red sage root liposome in the red sage root injection is 4wt%.
Comparative example 4
In contrast to example 3, only the acid treatment was included in step S3, and the other conditions were not changed.
The method specifically comprises the following steps:
s1, water extraction of the red sage root: decocting Saviae Miltiorrhizae radix with water at a mass ratio of Saviae Miltiorrhizae radix to water of 1:4g/mL for 1.5h for 3 times, filtering with 60 mesh sieve, mixing filtrates, and concentrating to relative density of 1.4 to obtain Saviae Miltiorrhizae radix water extract;
s2, alcohol precipitation: adding ethanol into the water extract of the red sage root prepared in the step S1 until the ethanol content in the system is 70%, performing first precipitation for 13h, filtering, continuously adding ethanol into the filtrate until the ethanol content in the system is 80%, performing second precipitation for 4h, filtering, and performing reduced pressure distillation on the filtrate to recover ethanol to obtain an ethanol precipitation solution;
s3, acid treatment: regulating the pH value of the alcohol precipitation solution obtained in the step S2 to 6 by using 2mol/L HCl solution, heating and boiling for 1h, cooling to room temperature, and filtering to obtain filtrate;
s4, decoloring: adding 8 parts by weight of activated carbon into 100 parts by weight of the filtrate obtained in the step S3, heating to 45 ℃, stirring for 25min, and filtering to obtain decolorized solution;
s5, ultrafiltration: ultrafiltering the decolorized solution obtained in the step S4 with 4000D ultrafiltration membrane with aperture for 45min to obtain ultrafiltrate;
s6, preparing salvia miltiorrhiza liposome: dissolving 4 parts by weight of egg yolk lecithin and 6 parts by weight of cholesterol in 60 parts by weight of a mixed solvent, wherein the mixed solvent is a mixture of dichloromethane and ethanol, the volume ratio is 2:5, dropwise adding 22 parts by weight of the ultrafiltrate prepared in the step S5, stirring and reacting for 2.5 hours after the dropwise adding is finished to obtain a liposome solution, removing the solvent under reduced pressure, freeze-drying, adding 100 parts by weight of a phosphate buffer solution with the pH value of 7.3, performing 1000W ultrasonic dispersion for 30min to obtain a liposome suspension, and preserving at 2 ℃;
S7, preparing polydopamine particles: dissolving 1.5 parts by weight of dopamine hydrochloride in 20 parts by weight of water, adding 30 parts by weight of ethanol and 5 parts by weight of 27wt% ammonia water, stirring for reaction for 22min at 1350r/min, centrifuging for 15min at 5000r/min, washing precipitate with deionized water, and freeze-drying to obtain polydopamine nanoparticles;
s8, preparing polydopamine-red sage root liposome: regulating the pH value of the salvia miltiorrhiza liposome suspension prepared in the step S6 to be 4.5, adding 1.5 parts by weight of polydopamine nanoparticles prepared in the step S7 into 10 parts by weight of the acidified salvia miltiorrhiza liposome suspension, stirring and reacting for 6 hours in a dark place, centrifuging for 15 minutes at 5000r/min, washing the precipitate with deionized water, and freeze-drying to obtain polydopamine-salvia miltiorrhiza liposome;
s9, preparing targeted peptide-polydopamine-red sage root liposome: dispersing 10 parts by weight of polydopamine-red sage root liposome in 50 parts by weight of water, adding 2.5 parts by weight of fibrin targeting peptide, uniformly mixing, stirring for reacting for 20 hours, and freeze-drying to obtain the targeting peptide-polydopamine-red sage root liposome;
the fibrin targeting peptide is a mixture of CREKA lipopeptide and GPRPP lipopeptide, and the mass ratio is 4:1;
s10, preparing a red sage root injection: adding the targeting peptide-polydopamine-red sage root liposome prepared in the step S9 into water for injection, regulating the pH value to 6.8, stirring, uniformly mixing, filling, sealing and sterilizing to obtain the red sage root injection, wherein the content of the targeting peptide-polydopamine-red sage root liposome in the red sage root injection is 4wt%.
Comparative example 5
In contrast to example 3, only the alkali treatment was included in step S3, and the other conditions were not changed.
The method specifically comprises the following steps:
s1, water extraction of the red sage root: decocting Saviae Miltiorrhizae radix with water at a mass ratio of Saviae Miltiorrhizae radix to water of 1:4g/mL for 1.5h for 3 times, filtering with 60 mesh sieve, mixing filtrates, and concentrating to relative density of 1.4 to obtain Saviae Miltiorrhizae radix water extract;
s2, alcohol precipitation: adding ethanol into the water extract of the red sage root prepared in the step S1 until the ethanol content in the system is 70%, performing first precipitation for 13h, filtering, continuously adding ethanol into the filtrate until the ethanol content in the system is 80%, performing second precipitation for 4h, filtering, and performing reduced pressure distillation on the filtrate to recover ethanol to obtain an ethanol precipitation solution;
s3, alkali treatment: regulating the pH value of the alcohol precipitation solution obtained in the step S2 to 9 by using a 2mol/L NaOH solution, heating and boiling for 1.5h, cooling to room temperature, and filtering to obtain filtrate;
s4, decoloring: adding 8 parts by weight of activated carbon into 100 parts by weight of the filtrate obtained in the step S3, heating to 45 ℃, stirring for 25min, and filtering to obtain decolorized solution;
s5, ultrafiltration: ultrafiltering the decolorized solution obtained in the step S4 with 4000D ultrafiltration membrane with aperture for 45min to obtain ultrafiltrate;
s6, preparing salvia miltiorrhiza liposome: dissolving 4 parts by weight of egg yolk lecithin and 6 parts by weight of cholesterol in 60 parts by weight of a mixed solvent, wherein the mixed solvent is a mixture of dichloromethane and ethanol, the volume ratio is 2:5, dropwise adding 22 parts by weight of the ultrafiltrate prepared in the step S5, stirring and reacting for 2.5 hours after the dropwise adding is finished to obtain a liposome solution, removing the solvent under reduced pressure, freeze-drying, adding 100 parts by weight of a phosphate buffer solution with the pH value of 7.3, performing 1000W ultrasonic dispersion for 30min to obtain a liposome suspension, and preserving at 2 ℃;
S7, preparing polydopamine particles: dissolving 1.5 parts by weight of dopamine hydrochloride in 20 parts by weight of water, adding 30 parts by weight of ethanol and 5 parts by weight of 27wt% ammonia water, stirring for reaction for 22min at 1350r/min, centrifuging for 15min at 5000r/min, washing precipitate with deionized water, and freeze-drying to obtain polydopamine nanoparticles;
s8, preparing polydopamine-red sage root liposome: regulating the pH value of the salvia miltiorrhiza liposome suspension prepared in the step S6 to be 4.5, adding 1.5 parts by weight of polydopamine nanoparticles prepared in the step S7 into 10 parts by weight of the acidified salvia miltiorrhiza liposome suspension, stirring and reacting for 6 hours in a dark place, centrifuging for 15 minutes at 5000r/min, washing the precipitate with deionized water, and freeze-drying to obtain polydopamine-salvia miltiorrhiza liposome;
s9, preparing targeted peptide-polydopamine-red sage root liposome: dispersing 10 parts by weight of polydopamine-red sage root liposome in 50 parts by weight of water, adding 2.5 parts by weight of fibrin targeting peptide, uniformly mixing, stirring for reacting for 20 hours, and freeze-drying to obtain the targeting peptide-polydopamine-red sage root liposome;
the fibrin targeting peptide is a mixture of CREKA lipopeptide and GPRPP lipopeptide, and the mass ratio is 4:1;
s10, preparing a red sage root injection: adding the targeting peptide-polydopamine-red sage root liposome prepared in the step S9 into water for injection, regulating the pH value to 6.8, stirring, uniformly mixing, filling, sealing and sterilizing to obtain the red sage root injection, wherein the content of the targeting peptide-polydopamine-red sage root liposome in the red sage root injection is 4wt%.
Comparative example 6
In comparison with example 3, the acid-base treatment of step S3 was not performed, and the other conditions were not changed.
The method specifically comprises the following steps:
s1, water extraction of the red sage root: decocting Saviae Miltiorrhizae radix with water at a mass ratio of Saviae Miltiorrhizae radix to water of 1:4g/mL for 1.5h for 3 times, filtering with 60 mesh sieve, mixing filtrates, and concentrating to relative density of 1.4 to obtain Saviae Miltiorrhizae radix water extract;
s2, alcohol precipitation: adding ethanol into the water extract of the red sage root prepared in the step S1 until the ethanol content in the system is 70%, performing first precipitation for 13h, filtering, continuously adding ethanol into the filtrate until the ethanol content in the system is 80%, performing second precipitation for 4h, filtering, and performing reduced pressure distillation on the filtrate to recover ethanol to obtain an ethanol precipitation solution;
s3, decoloring: adding 8 parts by weight of activated carbon into 100 parts by weight of the alcohol precipitation solution prepared in the step S2, heating to 45 ℃, stirring for 25min, and filtering to obtain decolorized solution;
s4, ultrafiltration: ultrafiltering the decolorized solution obtained in the step S3 with 4000D ultrafiltration membrane with aperture for 45min to obtain ultrafiltrate;
s5, preparing salvia miltiorrhiza liposome: dissolving 4 parts by weight of egg yolk lecithin and 6 parts by weight of cholesterol in 60 parts by weight of a mixed solvent, wherein the mixed solvent is a mixture of dichloromethane and ethanol, the volume ratio is 2:5, dropwise adding 22 parts by weight of the ultrafiltrate prepared in the step S4, stirring and reacting for 2.5 hours after the dropwise adding is finished to obtain a liposome solution, removing the solvent under reduced pressure, freeze-drying, adding 100 parts by weight of a phosphate buffer solution with the pH value of 7.3, performing 1000W ultrasonic dispersion for 30min to obtain a liposome suspension, and preserving at 2 ℃;
S6, preparing polydopamine particles: dissolving 1.5 parts by weight of dopamine hydrochloride in 20 parts by weight of water, adding 30 parts by weight of ethanol and 5 parts by weight of 27wt% ammonia water, stirring for reaction for 22min at 1350r/min, centrifuging for 15min at 5000r/min, washing precipitate with deionized water, and freeze-drying to obtain polydopamine nanoparticles;
s7, preparing polydopamine-red sage root liposome: regulating the pH value of the salvia miltiorrhiza liposome suspension prepared in the step S5 to be 4.5, adding 1.5 parts by weight of polydopamine nanoparticles prepared in the step S6 into 10 parts by weight of the acidified salvia miltiorrhiza liposome suspension, stirring and reacting for 6 hours in a dark place, centrifuging for 15 minutes at 5000r/min, washing the precipitate with deionized water, and freeze-drying to obtain polydopamine-salvia miltiorrhiza liposome;
s8, preparing targeted peptide-polydopamine-red sage root liposome: dispersing 10 parts by weight of polydopamine-red sage root liposome in 50 parts by weight of water, adding 2.5 parts by weight of fibrin targeting peptide, uniformly mixing, stirring for reacting for 20 hours, and freeze-drying to obtain the targeting peptide-polydopamine-red sage root liposome;
the fibrin targeting peptide is a mixture of CREKA lipopeptide and GPRPP lipopeptide, and the mass ratio is 4:1;
s9, preparing a red sage root injection: adding the targeting peptide-polydopamine-red sage root liposome prepared in the step S8 into water for injection, regulating the pH value to 6.8, stirring, uniformly mixing, filling, sealing and sterilizing to obtain the red sage root injection, wherein the content of the targeting peptide-polydopamine-red sage root liposome in the red sage root injection is 4wt%.
Comparative example 7
In contrast to example 3, steps S2 to S4 were not performed, and the other conditions were not changed.
The method specifically comprises the following steps:
s1, water extraction of the red sage root: decocting Saviae Miltiorrhizae radix with water at a mass ratio of Saviae Miltiorrhizae radix to water of 1:4g/mL for 1.5h for 3 times, filtering with 60 mesh sieve, mixing filtrates, and concentrating to relative density of 1.4 to obtain Saviae Miltiorrhizae radix water extract;
s2, ultrafiltration: ultrafiltering the Saviae Miltiorrhizae radix water extract obtained in step S1 with 4000D ultrafiltration membrane for 45min to obtain ultrafiltrate;
s3, preparing salvia miltiorrhiza liposome: dissolving 4 parts by weight of egg yolk lecithin and 6 parts by weight of cholesterol in 60 parts by weight of a mixed solvent, wherein the mixed solvent is a mixture of dichloromethane and ethanol, the volume ratio is 2:5, dropwise adding 22 parts by weight of the ultrafiltrate prepared in the step S2, stirring and reacting for 2.5 hours after the dropwise adding is finished to obtain a liposome solution, removing the solvent under reduced pressure, freeze-drying, adding 100 parts by weight of a phosphate buffer solution with the pH value of 7.3, performing 1000W ultrasonic dispersion for 30min to obtain a liposome suspension, and preserving at 2 ℃;
s4, preparing polydopamine particles: dissolving 1.5 parts by weight of dopamine hydrochloride in 20 parts by weight of water, adding 30 parts by weight of ethanol and 5 parts by weight of 27wt% ammonia water, stirring for reaction for 22min at 1350r/min, centrifuging for 15min at 5000r/min, washing precipitate with deionized water, and freeze-drying to obtain polydopamine nanoparticles;
S5, preparing polydopamine-red sage root liposome: regulating the pH value of the salvia miltiorrhiza liposome suspension prepared in the step S3 to be 4.5, adding 1.5 parts by weight of polydopamine nanoparticles prepared in the step S4 into 10 parts by weight of the acidified salvia miltiorrhiza liposome suspension, stirring and reacting for 6 hours in a dark place, centrifuging for 15 minutes at 5000r/min, washing the precipitate with deionized water, and freeze-drying to obtain polydopamine-salvia miltiorrhiza liposome;
s6, preparing targeted peptide-polydopamine-red sage root liposome: dispersing 10 parts by weight of polydopamine-red sage root liposome in 50 parts by weight of water, adding 2.5 parts by weight of fibrin targeting peptide, uniformly mixing, stirring for reacting for 20 hours, and freeze-drying to obtain the targeting peptide-polydopamine-red sage root liposome;
the fibrin targeting peptide is a mixture of CREKA lipopeptide and GPRPP lipopeptide, and the mass ratio is 4:1;
s7, preparing a red sage root injection: adding the targeting peptide-polydopamine-red sage root liposome prepared in the step S6 into water for injection, regulating the pH value to 6.8, stirring, uniformly mixing, filling, sealing and sterilizing to obtain the red sage root injection, wherein the content of the targeting peptide-polydopamine-red sage root liposome in the red sage root injection is 4wt%.
Comparative example 8
In contrast to example 3, step S6 was not performed, and the other conditions were not changed.
The method specifically comprises the following steps:
s1, water extraction of the red sage root: decocting Saviae Miltiorrhizae radix with water at a mass ratio of Saviae Miltiorrhizae radix to water of 1:4g/mL for 1.5h for 3 times, filtering with 60 mesh sieve, mixing filtrates, and concentrating to relative density of 1.4 to obtain Saviae Miltiorrhizae radix water extract;
s2, alcohol precipitation: adding ethanol into the water extract of the red sage root prepared in the step S1 until the ethanol content in the system is 70%, performing first precipitation for 13h, filtering, continuously adding ethanol into the filtrate until the ethanol content in the system is 80%, performing second precipitation for 4h, filtering, and performing reduced pressure distillation on the filtrate to recover ethanol to obtain an ethanol precipitation solution;
s3, acid-base treatment: regulating the pH value of the alcohol precipitation solution obtained in the step S2 to 9 by using 2mol/L NaOH solution, heating and boiling for 1.5h, cooling to room temperature, filtering, regulating the pH value of the filtrate to 6 by using 2mol/L HCl solution, heating and boiling for 1h, cooling to room temperature, and filtering to obtain filtrate;
s4, decoloring: adding 8 parts by weight of activated carbon into 100 parts by weight of the filtrate obtained in the step S3, heating to 45 ℃, stirring for 25min, and filtering to obtain decolorized solution;
s5, ultrafiltration: ultrafiltering the decolorized solution obtained in the step S4 with 4000D ultrafiltration membrane with aperture for 45min to obtain ultrafiltrate;
s6, preparing polydopamine particles: dissolving 1.5 parts by weight of dopamine hydrochloride in 20 parts by weight of water, adding 30 parts by weight of ethanol and 5 parts by weight of 27wt% ammonia water, stirring for reaction for 22min at 1350r/min, centrifuging for 15min at 5000r/min, washing precipitate with deionized water, and freeze-drying to obtain polydopamine nanoparticles;
S7, preparing polydopamine-red sage root: regulating the pH value of the ultrafiltrate obtained in the step S5 to be 4.5, adding 1.5 parts by weight of polydopamine nanoparticles obtained in the step S7 into 10 parts by weight of the acidified ultrafiltrate, stirring and reacting for 6 hours in a dark place, centrifuging for 15 minutes at 5000r/min, washing the precipitate with deionized water, and freeze-drying to obtain polydopamine-red sage root;
s8, preparing a targeting peptide-polydopamine-red sage root: dispersing 10 parts by weight of polydopamine-red sage root in 50 parts by weight of water, adding 2.5 parts by weight of fibrin targeting peptide, uniformly mixing, stirring for reacting for 20 hours, and freeze-drying to obtain the targeting peptide polydopamine-red sage root;
the fibrin targeting peptide is a mixture of CREKA lipopeptide and GPRPP lipopeptide, and the mass ratio is 4:1;
s9, preparing a red sage root injection: adding the targeting peptide-polydopamine-red sage root prepared in the step S8 into water for injection, regulating the pH value to 6.8, stirring and mixing uniformly, filling and sealing, and sterilizing to obtain the red sage root injection, wherein the content of the targeting peptide-polydopamine-red sage root in the red sage root injection is 4wt%.
Comparative example 9
In contrast to example 3, steps S7 to S8 were not performed, and the other conditions were not changed.
The method specifically comprises the following steps:
s1, water extraction of the red sage root: decocting Saviae Miltiorrhizae radix with water at a mass ratio of Saviae Miltiorrhizae radix to water of 1:4g/mL for 1.5h for 3 times, filtering with 60 mesh sieve, mixing filtrates, and concentrating to relative density of 1.4 to obtain Saviae Miltiorrhizae radix water extract;
S2, alcohol precipitation: adding ethanol into the water extract of the red sage root prepared in the step S1 until the ethanol content in the system is 70%, performing first precipitation for 13h, filtering, continuously adding ethanol into the filtrate until the ethanol content in the system is 80%, performing second precipitation for 4h, filtering, and performing reduced pressure distillation on the filtrate to recover ethanol to obtain an ethanol precipitation solution;
s3, acid-base treatment: regulating the pH value of the alcohol precipitation solution obtained in the step S2 to 9 by using 2mol/L NaOH solution, heating and boiling for 1.5h, cooling to room temperature, filtering, regulating the pH value of the filtrate to 6 by using 2mol/L HCl solution, heating and boiling for 1h, cooling to room temperature, and filtering to obtain filtrate;
s4, decoloring: adding 8 parts by weight of activated carbon into 100 parts by weight of the filtrate obtained in the step S3, heating to 45 ℃, stirring for 25min, and filtering to obtain decolorized solution;
s5, ultrafiltration: ultrafiltering the decolorized solution obtained in the step S4 with 4000D ultrafiltration membrane with aperture for 45min to obtain ultrafiltrate;
s6, preparing salvia miltiorrhiza liposome: dissolving 4 parts by weight of egg yolk lecithin and 6 parts by weight of cholesterol in 60 parts by weight of a mixed solvent, wherein the mixed solvent is a mixture of dichloromethane and ethanol, the volume ratio is 2:5, dropwise adding 22 parts by weight of the ultrafiltrate prepared in the step S5, stirring and reacting for 2.5 hours after the dropwise adding is finished to obtain a liposome solution, removing the solvent under reduced pressure, freeze-drying, adding 100 parts by weight of a phosphate buffer solution with the pH value of 7.3, performing 1000W ultrasonic dispersion for 30min to obtain a liposome suspension, and preserving at 2 ℃;
S7, preparing targeting peptide-salvia miltiorrhiza liposome: adding 2.5 parts by weight of fibrin targeting peptide into 60 parts by weight of liposome suspension prepared in the step S6, uniformly mixing, stirring and reacting for 20 hours, and freeze-drying to obtain targeting peptide-red sage root liposome;
the fibrin targeting peptide is a mixture of CREKA lipopeptide and GPRPP lipopeptide, and the mass ratio is 4:1;
s8, preparing a red sage root injection: adding the targeting peptide-red sage root liposome prepared in the step S7 into water for injection, regulating the pH value to 6.8, stirring and mixing uniformly, encapsulating and sterilizing to obtain the red sage root injection, wherein the content of the targeting peptide-red sage root liposome in the red sage root injection is 4wt%.
Comparative example 10
In contrast to example 3, step S9 was not performed, and the other conditions were not changed.
The method specifically comprises the following steps:
s1, water extraction of the red sage root: decocting Saviae Miltiorrhizae radix with water at a mass ratio of Saviae Miltiorrhizae radix to water of 1:4g/mL for 1.5h for 3 times, filtering with 60 mesh sieve, mixing filtrates, and concentrating to relative density of 1.4 to obtain Saviae Miltiorrhizae radix water extract;
s2, alcohol precipitation: adding ethanol into the water extract of the red sage root prepared in the step S1 until the ethanol content in the system is 70%, performing first precipitation for 13h, filtering, continuously adding ethanol into the filtrate until the ethanol content in the system is 80%, performing second precipitation for 4h, filtering, and performing reduced pressure distillation on the filtrate to recover ethanol to obtain an ethanol precipitation solution;
S3, acid-base treatment: regulating the pH value of the alcohol precipitation solution obtained in the step S2 to 9 by using 2mol/L NaOH solution, heating and boiling for 1.5h, cooling to room temperature, filtering, regulating the pH value of the filtrate to 6 by using 2mol/L HCl solution, heating and boiling for 1h, cooling to room temperature, and filtering to obtain filtrate;
s4, decoloring: adding 8 parts by weight of activated carbon into 100 parts by weight of the filtrate obtained in the step S3, heating to 45 ℃, stirring for 25min, and filtering to obtain decolorized solution;
s5, ultrafiltration: ultrafiltering the decolorized solution obtained in the step S4 with 4000D ultrafiltration membrane with aperture for 45min to obtain ultrafiltrate;
s6, preparing salvia miltiorrhiza liposome: dissolving 4 parts by weight of egg yolk lecithin and 6 parts by weight of cholesterol in 60 parts by weight of a mixed solvent, wherein the mixed solvent is a mixture of dichloromethane and ethanol, the volume ratio is 2:5, dropwise adding 22 parts by weight of the ultrafiltrate prepared in the step S5, stirring and reacting for 2.5 hours after the dropwise adding is finished to obtain a liposome solution, removing the solvent under reduced pressure, freeze-drying, adding 100 parts by weight of a phosphate buffer solution with the pH value of 7.3, performing 1000W ultrasonic dispersion for 30min to obtain a liposome suspension, and preserving at 2 ℃;
s7, preparing polydopamine particles: dissolving 1.5 parts by weight of dopamine hydrochloride in 20 parts by weight of water, adding 30 parts by weight of ethanol and 5 parts by weight of 27wt% ammonia water, stirring for reaction for 22min at 1350r/min, centrifuging for 15min at 5000r/min, washing precipitate with deionized water, and freeze-drying to obtain polydopamine nanoparticles;
S8, preparing polydopamine-red sage root liposome: regulating the pH value of the salvia miltiorrhiza liposome suspension prepared in the step S6 to be 4.5, adding 1.5 parts by weight of polydopamine nanoparticles prepared in the step S7 into 10 parts by weight of the acidified salvia miltiorrhiza liposome suspension, stirring and reacting for 6 hours in a dark place, centrifuging for 15 minutes at 5000r/min, washing the precipitate with deionized water, and freeze-drying to obtain polydopamine-salvia miltiorrhiza liposome;
s9, preparing a red sage root injection: adding the polydopamine-red sage liposome prepared in the step S8 into water for injection, regulating the pH value to 6.8, stirring and mixing uniformly, filling and sealing, and sterilizing to obtain the red sage root injection, wherein the content of polydopamine-red sage root liposome in the red sage root injection is 4wt%.
Test example 1
The content of sodium danshensu, protocatechuic aldehyde, salvianolic acid B and salvianolic acid A in the red sage root injection of examples 1-5 and comparative examples 1-10 was measured by HPLC method, and the results are shown in Table 1.
Treating the red sage root injection, placing the prepared red sage root injection into an ultracentrifuge tube, centrifuging for 30min in a high-speed centrifuge at 18000r/min, and taking the supernatant for testing.
In the HPLC method detection:
control: sodium salvianolate (middle check), protocatechuic aldehyde (middle check), salvianolic acid B (middle check), and salvianolic acid A (homemade, normalized and calibrated content 98.5%).
Chromatographic conditions: chromatographic column: agilent1100 high performance liquid chromatography system, agilent ZORBAX 80AExtend-C18 column (4.6X1250 mm), mobile phase A: water-acetonitrile-formic acid (90:10:0.5, v/v), mobile phase B: acetonitrile; flow rate: 1.0mL/min, detection wavelength: 280nm, column temperature: 30 ℃, DAD scan range: 190-400nm. Linear gradient elution procedure used: 0% B,20min15% B,55min25% B,75min85% B,90min85B%.
TABLE 1
As shown in the table above, the content of each active ingredient in the red sage root injection prepared in the examples 1-3 of the invention meets the requirements, and the content of the active ingredient is higher.
Test example 2
The ultrafiltrates of examples 1-5 and comparative examples 1-10 were examined for their tannin removal effect. The results are shown in Table 2.
According to the method for detecting tannin under the injection item of the appendices of the Chinese pharmacopoeia of 2000 edition. Taking ultrafiltration liquid 1m1, adding newly prepared physiological saline containing 1% egg white 5m1, and standing for 10min without turbidity or precipitation. Or adding diluted acetic acid 1 drop into ultrafiltrate 1m1, and adding gelatin sodium chloride solution (gelatin 1%, 10% aqueous solution of sodium chloride, and new formulation for use) 4-5 drops to obtain turbidity and precipitate.
TABLE 2
Group of Effect of removing tanning Loss ratio (in terms of index component) (%)
Example 1 — 12.1
Example 2 — 11.3
Example 3 — 10.4
Example 4 — 13.2
Example 5 — 12.9
Comparative example 1 * 19.2
Comparative example 2 * 20.2
Comparative example 3 ** 24.5
Comparative example 4 ** 23.7
Comparative example 5 ** 24.9
Comparative example 6 *** 30.3
Comparative example 7 **** 45.8
Comparative example 8 — 17.4
Comparative example 9 — 16.5
Comparative example 10 — 14.2
Annotation: * ***: large-scale precipitation; * **: obvious turbidity; * *: cloudiness; * : slightly cloudy; -: clarifying.
As shown in the table above, the results of the tannins test of the salvia miltiorrhiza injection prepared in the examples 1-3 of the invention all meet the requirements, and the injection is clear.
Test example 3
Taking SPF grade 7-8 week old healthy male ApoE -/- The mice, weight of 17-19g, general feed adaptability feeding for 1 week, randomly selecting 8 as normal group, feeding with general feed, feeding the rest mice with high fat feed for 6 weeks, then dividing into model group, example 1-5 group, comparative example 1-10 group, atorvastatin group, 8 each, example 1-5 group, comparative example 1-10 group to corresponding red sage root injection 5mL/kg, atorvastatin group to atorvastatin 4mg/kg, model group to normal saline 5mL/kg, continuously administering for 8 weeks, killing mice.
Mice were collected from eyeballs, centrifuged, and the supernatant serum was collected and stored at-80 ℃. Total Cholesterol (TC) content was measured with a fully automatic biochemical analyzer. ELISA method for detecting the content of TNF-alpha and IL-6 in serum. Thiobarbituric acid (TBA) detects the content of MDA in serum. The total SOD activity detection kit (WST-8) detects serum SOD activity.
The results are shown in Table 3.
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Annotation: p <0.05 compared to normal group, # compared to model group, P <0.05.
As shown in the table above, the red sage root injection prepared in the examples 1-3 of the invention can obviously reduce the cholesterol content and inflammatory factor content of the high-fat mice and improve the antioxidant capacity.
Test example 4 thrombolytic mouse test
Taking 8-10 week old healthy male mice, weighing 22+ -2 g, randomly dividing into a control group, an aspirin group, an example 1-5 group and a comparative example 1-10 group, wherein 8 animals in each group are sterilized and high in nutrition, and adding 3-4d of feed each time, and adding cooked eggs and sterilized sunflower seeds. Examples 1-5 and comparative examples 1-10 mice were injected with 2mL/kg of the corresponding prepared red sage root injection daily, and the control group was given an equivalent amount of physiological saline, and the aspirin group was given 100mg/kg of aspirin. After 3d of administration, each group was intraperitoneally injected with carrageenan (20 mg/kg,10 mL/kg) and continued for 2d, and the black tail rate and black tail length after 12h, 24h, 48h of administration of carrageenan were measured and recorded, respectively. The results are shown in Table 4.
TABLE 4 Table 4
Annotation: p <0.05 compared to control group.
As shown in the table above, the red sage root injection prepared in the examples 1-3 of the present invention can obviously reduce the black tail rate and the black tail length of mice, and has obvious thrombolytic effect.
Examples 4 and 5 compared with example 3, the fibrin targeting peptide is a single CREKA lipopeptide or GPRPP lipopeptide, total cholesterol, inflammatory factors TNF- α, IL-6 are improved, antioxidant ability is reduced, black tail rate and black tail length are improved. Comparative example 10 compared to example 3, step S9 was not performed. Fibrin is a specific sign of thrombus, total cholesterol, inflammatory factors TNF-alpha and IL-6 are obviously improved, the antioxidant capacity is obviously reduced, and the black tail rate and the black tail length are obviously improved. During thrombosis, thrombin and coagulation factors catalyze the production of fibrin from fibrinogen, while fibrin cross-links to each other to form a dense biopolymer network, thereby protecting the blood clot from mechanical stress and proteolytic attack and activating platelet recruitment, which is known to be present in large amounts at the thrombus site. The lipopeptides CREKA and GPRPP are fibrin targeting lipopeptides, the CREKA is linear and only comprises 5 amino acid residues, in addition, the tail end amino acid-cysteine in the CREKA can be biologically coupled through thiol-maleimide reaction, so that the targeting effect is realized, meanwhile, 1-4 amino acids on the rear end chains of the two can be used as connectors, and the targeting effect of the lipopeptides is not influenced. The fibrin targeting peptide is a mixture of CREKA and GPRPP, is connected with the polydopamine-salvia miltiorrhiza liposome compound, and combines the two, under the synergistic effect, the targeting speed and accuracy are improved, so that the salvia miltiorrhiza liposome can realize efficient thrombus homing, the polydopamine-salvia miltiorrhiza liposome compound can be selectively released in the environment of thrombus, the rapid targeting drug release of the salvia miltiorrhiza extract is realized, and the excellent thrombolysis effect and the good biological safety are shown.
In comparative examples 1 and 2, only the first precipitation or the second precipitation was included in step S2, and the tanning effect was decreased, the loss was increased, and the content of each active was slightly decreased, as compared with example 3. Comparative example 3 compared with example 3, the step S2 alcohol precipitation step was not performed, the tanning effect was reduced, the loss was increased, and the content of each active material was reduced. The polysaccharide component in the red sage root water extract is removed through alcohol precipitation treatment, so that polysaccharide floccules in the later injection are avoided, and the quality of the injection is influenced.
In comparative examples 4 and 5, the acid treatment or the alkali treatment was only included in the step S3, the tanning effect was significantly reduced, the loss was increased, and the content of each active was slightly reduced, as compared with the example 3. Comparative example 6 compared with example 3, the acid-base treatment of step S3 was not performed, the tanning effect was significantly reduced, the loss was increased, and the content of each active material was significantly reduced. The invention can fully destroy tannins, proteins and unstable components in the red sage root extracting solution through acid-base treatment, and simultaneously improve the content of active ingredients of the red sage root extract.
Comparative example 7 compared with example 3, the tanning effect was significantly reduced without performing steps S2 to S4, the loss was significantly increased, and the content of each active was significantly reduced. The invention firstly removes a large amount of macromolecular polysaccharide, protein, tannin and pigment substances after alcohol precipitation, acid-base treatment and decoloration, and the remaining micromolecular active substances can be obviously purified after ultrafiltration, so that the ultrafiltration efficiency is high, and the obtained active compounds mostly are active compounds of the red sage root, and the ultrafiltration loss is reduced.
Comparative example 8 compared with example 3, the total cholesterol, inflammatory factors TNF-alpha, IL-6 were significantly improved, the antioxidant capacity was significantly reduced, and the black tail rate and black tail length were significantly improved without performing step S6. In the salvia miltiorrhiza injection prepared by the invention, active substances of the salvia miltiorrhiza comprise sodium danshensu, protocatechuic aldehyde, rosmarinic acid, salvianolic acid B, salvianolic acid A and the like, the active substances are wrapped in a liposome, and the hydroxyl on the surface of the liposome and the amino on the surface of polydopamine can be electrostatically adsorbed, so that a stable polydopamine-salvia miltiorrhiza liposome compound is formed, and a good sustained and controlled release effect is achieved. The active substances of the red sage root which are not embedded by the liposome are easy to escape, so that the effects of dissolving thrombus, reducing blood fat, resisting inflammation and resisting oxidation are not very good. In addition, the salvia miltiorrhiza bunge is mainly phenolic acid compounds which are extracted by water decoction, are unstable in heat and easy to decompose by heating, and the liposome embedded salvia miltiorrhiza bunge active substance has a better effect of stabilizing the salvia miltiorrhiza bunge active substance, so that the problems of medicine inactivation, drug effect reduction and the like caused by long-time storage are avoided, and the liposome embedded salvia miltiorrhiza bunge active substance has obvious practical significance.
Comparative example 9 compared with example 3, steps S7 to S8 were not performed, total cholesterol, inflammatory factors TNF- α, IL-6 were increased, antioxidant ability was decreased, and black tail rate and black tail length were increased. According to the invention, amino groups on the surface of polydopamine in polydopamine-salvia miltiorrhiza liposome can be electrostatically adsorbed with fibrin targeting peptides, such as carboxylic acid on lipopeptides CREKA and GPRPP, so that a stable targeting peptide-polydopamine-salvia miltiorrhiza liposome is formed, the prepared targeting peptide-polydopamine-salvia miltiorrhiza liposome can quickly and accurately directly reach thrombus focus under the action of the targeting peptide, the salvia miltiorrhiza liposome releases active substances of the salvia miltiorrhiza slowly, long-acting thrombolysis, antioxidation, vascular elasticity improvement and other effects are achieved, and the prepared salvia miltiorrhiza injection can achieve the effects of quickly, efficiently and effectively dissolving thrombus, relieving atherosclerosis, improving microcirculation, inhibiting platelet aggregation, resisting lipid peroxidation and the like. Without the connection of polydopamine, the combination of liposome and lipopeptide is unstable, thus the targeting drug delivery effect is not very good, and the thrombolysis, blood fat reduction, anti-inflammatory and antioxidant effects are reduced.
The foregoing description of the preferred embodiments of the invention is not intended to be limiting, but rather is intended to cover all modifications, equivalents, alternatives, and improvements that fall within the spirit and scope of the invention.

Claims (3)

1. The preparation method of the salvia miltiorrhiza injection is characterized by comprising the following steps of:
s1, water extraction of the red sage root: decocting Saviae Miltiorrhizae radix with water at a mass ratio of 1:3-5g/mL for 1-2h for 2-3 times, filtering, mixing filtrates, and concentrating to relative density of 1.3-1.5 to obtain Saviae Miltiorrhizae radix water extract;
s2, alcohol precipitation: adding ethanol into the water extract of radix Salviae Miltiorrhizae obtained in step S1 until the ethanol content is 65-75%, precipitating for 12-15 hr for the first time, filtering, adding ethanol into the filtrate until the ethanol content is 75-85%, precipitating for the second time for 3-5 hr, filtering, and distilling the filtrate under reduced pressure to recover ethanol to obtain ethanol precipitation solution;
s3, acid-base treatment: regulating pH of the ethanol precipitation solution obtained in step S2 to 8-10 with 1-3mol/L NaOH or KOH solution, heating and boiling for 1-2 hr, cooling, filtering, and filtering with 1-3mol/L HCl or H 2 SO 4 The pH value of the solution is regulated to 5-6.5, the solution is heated and boiled for 0.5-1h, cooled and filtered to obtain filtrate;
S4, decoloring: adding 5-12 parts by weight of activated carbon into 100 parts by weight of the filtrate obtained in the step S3, heating to 40-50 ℃, stirring for 20-30min, and filtering to obtain decolorized solution;
s5, ultrafiltration: ultrafiltering the decolorized solution obtained in the step S4 with an ultrafiltration membrane with a pore diameter of 3000-5000D for 30-60min to obtain ultrafiltrate;
s6, preparing salvia miltiorrhiza liposome: dissolving 3-5 parts by weight of egg yolk lecithin and 5-7 parts by weight of cholesterol in 50-70 parts by weight of mixed solvent, wherein the mixed solvent is a mixture of dichloromethane and ethanol, the volume ratio is 1-3:5, dropwise adding 20-25 parts by weight of the ultrafiltrate prepared in the step S5, stirring and reacting for 2-3 hours after the dropwise adding is finished to obtain a liposome solution, removing the solvent under reduced pressure, freeze-drying, adding 100 parts by weight of phosphate buffer with the pH value of 7.2-7.5, uniformly dispersing by ultrasonic waves to obtain a liposome suspension, and preserving at the temperature of 0-4 ℃;
s7, preparing polydopamine particles: dissolving 1-2 parts by weight of dopamine hydrochloride in 20 parts by weight of water, adding 20-40 parts by weight of ethanol and 3-7 parts by weight of 25-28wt% ammonia water, stirring and reacting for 15-30min at 1200-1500r/min, centrifuging, washing precipitate, and freeze-drying to obtain polydopamine nanoparticles;
s8, preparing polydopamine-red sage root liposome: regulating the pH value of the salvia miltiorrhiza liposome suspension prepared in the step S6 to be 4-5, adding 1-2 parts by weight of polydopamine nanoparticles prepared in the step S7 into 10 parts by weight of the acidified salvia miltiorrhiza liposome suspension, stirring and reacting for 5-7 hours in a dark place, centrifuging, washing the precipitate, and freeze-drying to obtain polydopamine-salvia miltiorrhiza liposome;
S9, preparing targeted peptide-polydopamine-red sage root liposome: dispersing 10 parts by weight of polydopamine-salvia miltiorrhiza liposome in water, adding 2-3 parts by weight of fibrin targeting peptide, uniformly mixing, stirring and reacting for 18-22 hours, and freeze-drying to obtain the targeting peptide-polydopamine-salvia miltiorrhiza liposome;
the fibrin targeting peptide is a mixture of CREKA lipopeptide and GPRPP lipopeptide, and the mass ratio is 3-5:1;
s10, preparing a red sage root injection: adding the targeting peptide-polydopamine-red sage liposome prepared in the step S9 into water for injection, regulating the pH value to 6.7-6.9, stirring and mixing uniformly, filling and sealing, and sterilizing to obtain the red sage root injection, wherein the content of the targeting peptide-polydopamine-red sage root liposome in the red sage root injection is 3.5-4.5wt%.
2. The method of claim 1.
3. Use of the red sage root injection according to claim 2 in the preparation of thrombolytic products.
CN202211153846.XA 2022-09-21 2022-09-21 Red sage root injection and its preparation method and application Active CN115429752B (en)

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