Improved nano suspension freeze-dried preparation based on perhexiline and preparation method thereof

文档序号：1329680 发布日期：2020-07-17 浏览：15次中文

阅读说明：本技术 一种基于冠心宁改进的纳米混悬冻干制剂及其制备方法 (Improved nano suspension freeze-dried preparation based on perhexiline and preparation method thereof ) 是由张继芬于 2020-05-20 设计创作，主要内容包括：本发明属于医药领域,涉及一种基于冠心宁改进的纳米混悬冻干制剂及其制备方法。本发明所解决的技术问题是提供一种同时含有川芎和丹参的难溶性和水溶性成分的口服制剂,解决了改善难溶性成分溶出,提高其口服生物利用度的技术问题。本发明的口服制剂是以中药材川芎和丹参的提取物为原料制成的口服冻干制剂：丹参提取物、川芎提取物的重量配比为8-12:6-10；制备方法如下：A、制备纳米混悬液：将丹参提取物、川芎提取物制成100-1000nm的纳米混悬液；B、将步骤A所得纳米混悬液冷冻干燥制成冻干粉。难溶性成分的表观溶解度较提取物显著提高,在体外模拟胃液和模拟肠液中的释放速率加快,累计释放度也显著增加,为临床应用川芎和丹参提供了一种新的技术思路。(The invention belongs to the field of medicines, and relates to an improved nano suspension freeze-dried preparation based on perhexiline and a preparation method thereof. The invention aims to provide an oral preparation containing insoluble and water-soluble components of ligusticum wallichii and salvia miltiorrhiza simultaneously, and solves the technical problems of improving the dissolution of the insoluble components and improving the oral bioavailability of the insoluble components. The oral preparation of the invention is an oral freeze-dried preparation prepared by taking extracts of traditional Chinese medicinal materials of ligusticum wallichii and salvia miltiorrhiza as raw materials: the weight ratio of the salvia miltiorrhiza extract to the ligusticum wallichii extract is 8-12: 6-10; the preparation method comprises the following steps: A. preparing a nano suspension: preparing the salvia miltiorrhiza extract and the ligusticum wallichii extract into 100-1000nm nanometer suspension; B. and D, freeze-drying the nano suspension obtained in the step A to prepare freeze-dried powder. The apparent solubility of the insoluble components is obviously improved compared with the extract, the release rate in-vitro simulated gastric juice and simulated intestinal juice is accelerated, the accumulated release degree is also obviously increased, and a new technical thought is provided for clinical application of the ligusticum wallichii and the salvia miltiorrhiza.)

1. The preparation method of the improved nano suspension freeze-dried preparation based on perhexiline is characterized by comprising the following steps: the oral freeze-dried preparation is prepared by taking a red sage root extract and a ligusticum wallichii extract as raw materials, wherein the ligusticum wallichii extract and the red sage root extract comprise the following components in percentage by weight:

the weight ratio of the salvia miltiorrhiza extract to the ligusticum wallichii extract is 8-12: 6-10; the preparation method comprises the following steps:

A. preparing a nano suspension: preparing the salvia miltiorrhiza extract and the ligusticum wallichii extract into 100-1000nm nanometer suspension;

B. and D, freeze-drying the nano suspension obtained in the step A to prepare freeze-dried powder.

2. The method for preparing the improved nano suspension freeze-dried preparation based on perhexiline according to claim 1, is characterized in that: the contents of the components of the ligusticum wallichii extract and the salvia miltiorrhiza extract are as follows:

3. the preparation method of the improved nano suspension freeze-dried preparation based on perhexiline according to the claim 1 or 2, characterized in that: the weight ratio of the salvia miltiorrhiza extract to the ligusticum wallichii extract is 10: 8.

4. The method for preparing the improved nano suspension freeze-dried preparation based on perhexiline according to claim 1, is characterized in that: the method for preparing the nano suspension in the step A comprises the following steps: an ultrasonic probe method after the precipitation of the anti-solvent, an anti-solvent precipitation method under water bath ultrasound, and an ultrasonic probe method after the precipitation of the anti-solvent under water bath ultrasound;

preferably, the method for preparing the nanosuspension in the step A adopts an ultrasonic method with a probe after anti-solvent precipitation under water bath ultrasound.

5. The preparation method of the improved nano suspension freeze-dried preparation based on perhexiline as claimed in claim 4, is characterized in that: step A, when preparing the nanometer suspension, adding a stabilizer into a water phase; the method for adding the stabilizer comprises the following steps: weighing stabilizer at a mass ratio of 1:0.25-1:1 of total amount of Saviae Miltiorrhizae radix extract and rhizoma Ligustici Chuanxiong extract to stabilizer, adding stabilizer into water, stirring and dissolving to obtain stabilizer water solution.

6. The method for preparing the improved nano suspension freeze-dried preparation based on perhexiline according to the claim 5, is characterized in that: the stabilizer is poloxamer188, polyvinylpyrrolidone-K30, Tween80, sodium dodecyl sulfate, and polyvinyl alcohol;

preferably, the stabilizer is tween80 or sodium dodecyl sulfate;

most preferably, the stabilizer is tween 80.

7. The method for preparing the improved nano suspension freeze-dried preparation based on perhexiline according to the claim 5, is characterized in that: at least one of the following is satisfied:

the addition amount of the stabilizer is the weight ratio of the total amount of the salvia miltiorrhiza extract and the ligusticum wallichii extract to the stabilizer is 1:0.25-1: 1;

preferably, Tween80 is used as a stabilizer, and the weight ratio of the total amount of the red sage root extract and the ligusticum wallichii extract to the stabilizer is 1:0.5-1: 1;

most preferably, Tween80 is used as a stabilizer, and the weight ratio of the total amount of the red sage root extract and the ligusticum wallichii extract to the stabilizer is 1: 1;

preferably, sodium dodecyl sulfate is used as a stabilizer, and the weight ratio of the total amount of the salvia miltiorrhiza extract and the ligusticum wallichii extract to the stabilizer is 1:0.25-1: 1;

most preferably, sodium dodecyl sulfate is used as a stabilizer, and the weight ratio of the total amount of the salvia miltiorrhiza extract and the ligusticum wallichii extract to the stabilizer is 1: 1.

8. The method for preparing the improved nano suspension freeze-dried preparation based on perhexiline according to claim 1, is characterized in that: at least one of the following is satisfied:

step B, adding a freeze-drying protective agent into the nano suspension, and then carrying out freeze drying;

preferably, the freeze-drying protective agent is maltose, mannitol, glucose, lactose;

most preferably, the lyoprotectant is maltose, mannitol, glucose;

preferably, the addition amount of the freeze-drying protective agent is 4-12g/100 ml;

preferably, the conditions of freeze-drying are as follows: pre-freezing at-40 deg.C to-20 deg.C for 2-8h, pre-freezing at-40 deg.C to-80 deg.C for 12-24h, and sublimation drying at 5-30 deg.C and under pressure lower than 15mTorr for 12-24 h;

most preferably, the conditions of freeze-drying are: prefreezing at-20 deg.C for 2 hr, prefreezing at-80 deg.C for 24 hr, and sublimation drying at 25 deg.C and pressure lower than 15mTorr for 24 hr.

9. The preparation method of the improved nano suspension freeze-dried preparation based on perhexiline as claimed in claim 4, is characterized in that: at least one of the following is satisfied:

adopting the ultrasonic method of the probe after the anti-solvent precipitation under the water bath ultrasonic:

1) weighing the salvia miltiorrhiza extract and the ligusticum wallichii extract according to the weight ratio to prepare the extract with the total concentration of 0.03-0.2 g.m L^-1Subjecting the 85-100% v/v ethanol solution to ultrasonic treatment for at least 10min, and centrifuging at 3000rpm or above for at least 10 min;

2) weighing the water phase and the centrifugal supernatant according to the volume ratio: the volume ratio of the water phase to the centrifugal supernatant is 5: 1-20: 1; placing the water phase in an ultrasonic water bath at the temperature of 0-15 ℃, rapidly adding the centrifugal supernatant under the stirring of 800-2000rpm and the ultrasonic of 200-500W water bath, and dissolving the centrifugal precipitate with a proper amount of pure water and then adding the dissolved centrifugal precipitate;

3) continuing ultrasonic stirring for 30-60min, connecting with a pressure reducing device, reducing pressure to-0.09 Mpa, volatilizing ethanol under water bath ultrasonic condition until no ethanol smell is evident, and performing ultrasonic treatment on the obtained suspension with a probe under ultrasonic power of 800-2000W for at least 3 min;

preferably, the ethanol solution in the step 1) in the probe ultrasonic method is 90% v/v after the anti-solvent precipitation under the water bath ultrasonic;

preferably, the magnetic stirring in step 2) of the probe ultrasonic method after the precipitation of the anti-solvent under the water bath ultrasonic is 1000-1200rpm, and the water bath ultrasonic is 300W;

preferably, after the anti-solvent is precipitated under the water bath ultrasound, the ultrasonic power in the step 3) of the probe ultrasonic method is preferably 1400-1800W; the ultrasonic treatment time is preferably 5-7 min;

adopting an anti-solvent precipitation method under water bath ultrasound:

1) weighing the salvia miltiorrhiza extract and the ligusticum wallichii extract according to the weight ratio to prepare the extract with the total concentration of 0.03-0.2 g.m L^-1Subjecting 85-100% v/v ethanol solution to ultrasonic treatment for at least 10min, and centrifuging at 3000rpm for at least 10 min;

3) continuing ultrasonic stirring for 30-60min, connecting with a pressure reducer, reducing pressure to-0.09 Mpa, and volatilizing ethanol in water bath ultrasonic state until no ethanol smell is observed;

preferably, the ethanol solution in the step 1) in the water bath ultrasonic precipitation method is 90% v/v;

preferably, the magnetic stirring in the step 2) of the probe ultrasonic method after the anti-solvent precipitation is 1000-1200rpm, and the water bath ultrasonic is 300W;

adopting an ultrasonic method of a probe after anti-solvent precipitation:

1) weighing the salvia miltiorrhiza extract and the ligusticum wallichii extract according to the weight ratio to prepare the extract with the total concentration of 0.03-0.2 g.m L^-1Subjecting 85-100% v/v ethanol solution to ultrasonic treatment for at least 10min, and centrifuging at 3000rpm for at least 10 min;

2) weighing the water phase and the centrifugal supernatant according to the volume ratio: the volume ratio of the water phase to the centrifugal supernatant is 5: 1-20: 1; rapidly adding the centrifugal supernatant into the water phase under the magnetic stirring of 800-;

preferably, the ethanol solution in the step 1) in the probe ultrasonic method after the anti-solvent precipitation is 90% v/v;

preferably, the magnetic stirring in the step 2) of the probe ultrasonic method after the anti-solvent precipitation is 1000-1200 rpm;

preferably, the ultrasonic power in the step 3) of the probe ultrasonic method after the antisolvent precipitation is preferably 1400-1800W; the ultrasound time is preferably 5-7 min.

10. An improved nano suspension freeze-dried preparation based on perhexiline prepared by the preparation method of any one of claims 1 to 9;

preferably, more than 90% of senkyunolide A, ligustilide, butenyl phthalide, cryptotanshinone, tanshinone I and tanshinone IIA in the nanometer suspension have particle size distribution less than 400 nm.

Technical Field

The invention belongs to the field of medicines, and relates to an improved nano suspension freeze-dried preparation based on perhexiline and a preparation method thereof.

Background

The perhexiline is a preparation prepared by taking ligusticum wallichii and salvia miltiorrhiza as raw materials according to the weight ratio of 1:1, and the existing preparation formulations on the market are tablets and injections. Guanxinning has the functions of promoting blood circulation to disperse blood clots, dredging meridian and nourishing heart, and is used mainly in treating coronary heart disease and angina pectoris.

At present, the research on the single medicinal components and pharmacological action of the ligusticum wallichii and the salvia miltiorrhiza shows that: the main alkaloid (ligustrazine) of rhizoma Ligustici Chuanxiong has effects of improving blood rheology, protecting coronary artery, etc., phenolic acid components (ligustrazine, ferulic acid, etc.) has effects of resisting blood platelet accumulation, scavenging oxygen free radical, dilating blood vessel, etc., and volatile oil (ligustilide, senkyunolide A, senkyunolide I, senkyunolide H, etc.) has pharmacological effects of relieving pain, tranquilizing, improving blood vessel function, protecting nerve cell, etc.; the chemical components of the salvia miltiorrhiza mainly comprise water-soluble phenolic acid compounds (salvianolic acids, protocatechuic aldehyde, protocatechuic acid, caffeic acid, rosmarinic acid, lithospermic acid and the like) and insoluble tanshinone compounds (tanshinone I, tanshinone IIA, tanshinone IIB, cryptotanshinone and the like), the water-soluble components have better antioxidation, anticoagulation, antithrombotic, blood fat regulation and cell protection effects, and the insoluble components have stronger effects of improving blood circulation, antibiosis, anti-inflammation and the like.

However, the existing medicinal materials of the coronary heart disease treating agent are processed by a water extraction process, the ingredients contained in the preparation mainly comprise danshensu, protocatechuic aldehyde, ligustrazine, senkyunolide I, senkyunolide H, rosmarinic acid, salvianolic acid B and the like, and insoluble ingredients such as senkyunolide A, ligustilide, tanshinone I, tanshinone IIA and the like which are beneficial to the clinical curative effect are not detected. The lack of the indissoluble drug effective components may reduce the clinical efficacy of perhexiline to a certain extent. Oral administration is the most common route of administration in clinical practice and is widely recognized for its advantages such as ease of administration, better patient compliance, etc. The present inventors have tried to prepare an oral preparation of perhexiline containing both poorly soluble and water soluble components, but have found that the poorly soluble components are limited in dissolution and are less absorbed by oral administration, and the pharmaceutical effects of these poorly soluble components cannot be exhibited even when prepared by a conventional method. For example, in the components of salvia miltiorrhiza and ligusticum wallichii which are commonly used for promoting blood circulation and removing blood stasis, the OB (Oralboavailability) values of the higher components tanshinone I, ligustilide and senkyunolide A are respectively only 29.27%, 23.50% and 26.56% (the data is from a TCMSP database), and are all lower than 30%, and 30% of the OB values are considered as the screening threshold value of the compound which can exert the drug effect. The low oral bioavailability of poorly soluble components has largely limited the clinical utility of these traditional Chinese medicines.

On the basis of the existing reports and application, the inventor of the invention provides a brand-new idea, and the oral nano freeze-dried preparation containing the insoluble components and the water-soluble components of the ligusticum wallichii and the salvia miltiorrhiza simultaneously is prepared by taking the ligusticum wallichii and the salvia miltiorrhiza as main raw materials, so that the defects of few types of active components and poor oral absorption of the insoluble active components of the existing preparation are overcome. In the existing reports, preparations prepared by using monomers thereof are also reported, such as the products on the market, i.e., salvianolate for injection, CN105496973A tanshinone IIA sodium sulfonate freeze-dried powder injection preparation and a preparation method thereof, and the freeze-drying method of the ligustrazine hydrochloride freeze-dried preparation for injection, CN105078907A freeze-dried powder injection and the like, but all the preparations are freeze-dried powder prepared by salifying and dissolving insoluble medicines in water, and no report related to the preparation of the freeze-dried preparation by using extracts is found, and no report related to the preparation of the insoluble component nano-suspension freeze-dried powder is found.

Disclosure of Invention

The invention aims to provide an oral preparation containing insoluble and water-soluble components of ligusticum wallichii and salvia miltiorrhiza simultaneously, and solves the technical problems of improving the dissolution of the insoluble components and improving the oral bioavailability of the insoluble components.

The oral preparation of the invention is an oral freeze-dried preparation prepared by taking extracts of traditional Chinese medicinal materials of ligusticum wallichii and salvia miltiorrhiza as raw materials: the weight ratio of the salvia miltiorrhiza extract to the ligusticum wallichii extract is 8-12: 6-10; preferably, the weight ratio of the salvia miltiorrhiza extract to the ligusticum wallichii extract is 10: 8.

The preparation method comprises the following steps:

A. preparing a nano suspension: preparing the salvia miltiorrhiza extract and the ligusticum wallichii extract into 100-1000nm nanometer suspension;

B. and D, freeze-drying the nano suspension obtained in the step A to prepare freeze-dried powder.

In the above technical scheme: the invention comprises the following components:

further preferably, the contents of the components of the ligusticum wallichii extract and the salvia miltiorrhiza extract are as follows:

the key to the preparation of nanoparticles of the present invention is the poorly soluble component: the key of the method is that the content of insoluble components in senkyunolide A, ligustilide, butenyl phthalide, cryptotanshinone, tanshinone I and tanshinone IIA influences whether nano-scale particles can be obtained: if the content of the insoluble component is too large, micron-sized particles are easily obtained, and the dissolution and bioavailability are influenced; however, the content of the insoluble component is too small, and nano-sized particles are easily formed, but effective drug loading cannot be realized, and the drug effect exertion is influenced.

In the above technical scheme: the method for preparing the nano suspension in the step A comprises the following steps: an ultrasonic probe method after the precipitation of the anti-solvent, an anti-solvent precipitation method under water bath ultrasound, and an ultrasonic probe method after the precipitation of the anti-solvent under water bath ultrasound. Preferably, the method adopts a probe ultrasonic method after anti-solvent precipitation under water bath ultrasonic.

Such as: adopting the ultrasonic method of the probe after the anti-solvent precipitation under the water bath ultrasonic:

preferably, the ethanol solution in the step 1) in the probe ultrasonic method is 90% v/v after the anti-solvent precipitation under the water bath ultrasonic;

Such as: adopting an anti-solvent precipitation method under water bath ultrasound:

1) weighing the salvia miltiorrhiza extract and the ligusticum wallichii extract according to the weight ratio to prepare the extract with the total concentration of 0.03-0.2 g.m L^-1Subjecting 85-100% v/v ethanol solution to ultrasonic treatment for at least 10min, and centrifuging at 3000rpm for at least 10 min;

preferably, the ethanol solution in the step 1) in the water bath ultrasonic precipitation method is 90% v/v;

preferably, the magnetic stirring in step 2) of the probe ultrasonic method after the antisolvent precipitation is 1000-1200rpm, and the water bath ultrasonic is 300W.

Such as: adopting an ultrasonic method of a probe after anti-solvent precipitation:

1) weighing the salvia miltiorrhiza extract and the ligusticum wallichii extract according to the weight ratio to prepare the extract with the total concentration of 0.03-0.2 g.m L^-1Subjecting 85-100% v/v ethanol solution to ultrasonic treatment for at least 10min, and centrifuging at 3000rpm for at least 10 min;

preferably, the ethanol solution in the step 1) in the probe ultrasonic method after the anti-solvent precipitation is 90% v/v;

preferably, the magnetic stirring in the step 2) of the probe ultrasonic method after the anti-solvent precipitation is 1000-1200 rpm;

preferably, the ultrasonic power in the step 3) of the probe ultrasonic method after the antisolvent precipitation is preferably 1400-1800W; the ultrasound time is preferably 5-7 min.

In the above technical scheme: when the nanosuspension is prepared in the step A, a stabilizing agent can be added into the water phase to ensure that the nanosuspension is maintained in a smaller particle size range for a longer time.

The method for adding the stabilizer comprises the following steps: weighing stabilizer at a mass ratio of 1:0.25-1:1 of total amount of Saviae Miltiorrhizae radix extract and rhizoma Ligustici Chuanxiong extract to stabilizer, adding stabilizer into water, stirring and dissolving to obtain stabilizer water solution.

Wherein, the stabilizer which can be added is any one of Poloxamer188, PVP K30, PVA, SDS and Tween 80; preferably the stabilizer is SDS or Tween 80; most preferably, the stabilizer is Tween 80.

Specifically, the addition amount of the stabilizer is that the weight ratio (drug-adjuvant ratio for short) of the total amount of the salvia miltiorrhiza extract and the ligusticum wallichii extract to the stabilizer is 1:0.25-1: 1. Wherein, the drug-adjuvant ratio of Tween80 used as a stabilizer is 1:0.5-1:1, and the preferred drug-adjuvant ratio is 1: 1. The drug-adjuvant ratio of SDS as stabilizer is 1:0.25-1:1, preferably 1: 1.

In the above technical scheme: and step B, adding a freeze-drying protective agent into the nano suspension, and then carrying out freeze drying.

Wherein the freeze-drying protective agent is maltose, mannitol, glucose and lactose. Preferably, maltose, mannitol, and glucose are used as lyoprotectants. The addition amount of the freeze-drying protective agent is 4-12g/100 ml.

Wherein the freeze-drying conditions are as follows: pre-freezing at-40 deg.C to-20 deg.C for 2-8h, pre-freezing at-40 deg.C to-80 deg.C for 12-24h, and sublimation drying at 5-30 deg.C and under pressure lower than 15mTorr for 12-24 h.

Preferred conditions for freeze-drying are: prefreezing at-20 deg.C for 2 hr, prefreezing at-80 deg.C for 24 hr, and sublimation drying at 25 deg.C and pressure lower than 15mTorr for 24 hr.

The method of the invention surpasses the current application situation that the prior Szechuan lovage rhizome and red sage root are either prepared into a preparation by adopting water extraction or alcohol extraction of crude medicinal materials of the Szechuan lovage rhizome and the red sage root or are prepared into a freeze-dried preparation by using water-soluble monomers or water-soluble derivatives of water-insoluble monomers. The nano suspension freeze-dried preparation containing both water-soluble active ingredients and insoluble active ingredients is obtained by utilizing the preparation method of firstly preparing the nano suspension and then freeze-drying the extract, the apparent solubility of the insoluble ingredients such as senkyunolide A, ligustilide, butylidene phthalide, cryptotanshinone, tanshinone I and tanshinone IIA is obviously improved compared with the extract, the release rate in vitro simulated gastric juice and simulated intestinal juice is accelerated, the accumulated release degree is also obviously increased, and a new technical idea is provided for clinical application of the ligusticum wallichii and the salvia miltiorrhiza.

Drawings

Figure 1 is an appearance diagram of each lyoprotectant.

Wherein: a is a sample before freeze-drying; b is a freeze-dried product; c is a redispersed sample; 1. 2 and 3 respectively represent the dosage of the cryoprotectant as 4 percent, 8 percent and 12 percent.

Fig. 2 shows the drug release curve (n ═ 3) of the perhexiline bulk drug and the nano suspension freeze-dried powder.

Detailed Description

The following description of specific embodiments of the invention illustrates, but does not limit, the invention.

In order to achieve the purpose that the nanosuspension freeze-dried preparation simultaneously contains water-soluble active ingredients and insoluble active ingredients, and the insoluble ingredients can be effectively released and dissolved, the inventor of the invention discovers that the perhexiline is subjected to subsequent process treatment by adopting an aqueous extract no matter the perhexiline is an oral preparation or an injection preparation, and the ingredients contained in the preparation mainly comprise danshensu, protocatechuic aldehyde, ligustrazine, senkyunolide I, senkyunolide H, rosmarinic acid, salvianolic acid B and the like, but the insoluble ingredients with specific efficacy reports, such as senkyunolide A, ligustilide, tanshinone I, tanshinone IIA and the like, are obviously deleted.

Aiming at insoluble components, various preparation technologies such as inclusion technology, solid dispersion technology, phospholipid complex, microemulsion and the like are used for improving the solubility and oral bioavailability of the insoluble components or medicines at present, but the preparation technologies all have certain defects, such as certain requirements of the inclusion technology on the structure of an included substance, low drug-loading rate of the solid dispersion technology, poor stability, easy aging and the like, and the problems of large dosage of a lipid carrier surfactant and certain potential safety hazard.

The nano suspension is a multiphase dispersion system with the grain diameter of 10-1000 nm, and the medicine is dispersed in a crystalline or amorphous state. The nanometer suspension can obviously improve the solubility of insoluble components and improve the oral bioavailability, and is successfully applied to a plurality of chemical pharmaceutical preparations. The application of the existing nano suspension technology in the field of traditional Chinese medicines still mainly takes traditional Chinese medicine monomers as main materials, such as: the existing research adopts the techniques of anti-solvent method, high-pressure homogenization, medium grinding, various combination techniques and the like to successfully prepare nano suspension of traditional Chinese medicine monomers such as curcumin, resveratrol, naringenin, silymarin, soybean isoflavone, magnolol, taxol, quercetin and the like; there are also a few monomer compositions and application research reports of effective parts, such as ciprofloxacin-catechin, curcumin-docetaxel, bilobalide, scutellaria total flavone, and nanometer suspension of herpetospermum pedunculosum total lignan. However, the clinical medicine of traditional Chinese medicine is characterized by the compatibility of the components, and 1 or 2 monomers or effective parts can not embody the comprehensive curative effect of the whole compound with multiple components, multiple ways and multiple targets. Compared with monomer components with definite structures or effective parts with similar chemical structures, the traditional Chinese medicine compound is a complex multi-component system, usually contains dozens or even hundreds of drug effect components, and the difference of the physicochemical properties of the components, such as solubility, pKa, stability and the like, is large; meanwhile, due to the existence of macromolecular ineffective components such as protein, polysaccharide, resin, mucilaginous substances and the like, the content of effective components in the compound extract is very low. The diversity, complexity and low content of active ingredients make the application of the nanosuspension technology in the traditional Chinese medicine compound difficult. At present, no application research of a nano suspension technology based on a compound multi-component system of traditional Chinese medicines exists, and the inventor tries to prepare a nano suspension system containing complex multi-components which are insoluble and water-soluble simultaneously on the application background; meanwhile, freeze-dried preparations prepared in the field of traditional Chinese medicines are also common in traditional Chinese medicine monomers, and no nano suspension freeze-dried preparation containing insoluble and water-soluble complex multiple components is available.

①, although the existing nano suspension preparation technology is more, but basically used for monomer components, compound preparation research generally takes extract as raw material, but the properties of the extract, such as solubility, viscosity and the like, are different from monomers, and whether the existing preparation method is suitable for raw material medicines of traditional Chinese medicine extract.

The raw material medicaments applied by the invention are salvia miltiorrhiza extract and ligusticum wallichii extract. The composition analysis of each extract is shown in table 1:

TABLE 1 content of each component in Salvia miltiorrhiza Bunge extract, Ligusticum chuanxiong Hort extract and Guanxinning tablet (n-3)/mg g^-1

-: not detected.

Analysis and conclusion: the original prescription of Guanxinning is prepared by 1:1 compatibility decoction of rhizoma ligustici wallichii and salvia miltiorrhiza, the salvia miltiorrhiza and rhizoma ligustici wallichii extracts used in the research are 80% alcohol extracts, the content of each component is different from the decoction of water, the extract yields of 2 medicinal materials after extraction are different, if 1:1 compatibility is still used, the subsequent researches of in vitro dissolution, in vivo bioavailability and the like all use Guanxinning tablets as reference, the detectable components of the Guanxinning tablets are used as reference, and the adopted commercially available salvia miltiorrhiza extract and rhizoma ligustici wallichii extract are used in a compatibility ratio of 10:8 by weight.

The following is a study of the method of preparation of the nanosuspension lyophilized formulation of the present invention.

Method and process selection

1. The following 7 preparation methods were compared, and the preparation methods were as follows:

(1) the anti-solvent precipitation method comprises collecting 0.1g Saviae Miltiorrhizae radix extract and 0.08g rhizoma Ligustici Chuanxiong extract, adding 5m L90% ethanol, ultrasonic treating for 15min, centrifuging at 5000rpm for 10min, rapidly adding the supernatant into 50m L pure water under magnetic stirring at 1000rpm, dissolving the precipitate with 1m L pure water, stirring for 30min, connecting a pressure reducer, and removing ethanol under reduced pressure (pressure less than-0.09 Mpa) for 1 hr.

(2) High pressure homogenizing method comprises weighing Saviae Miltiorrhizae radix extract 0.1g and rhizoma Ligustici Chuanxiong extract 0.08g, adding pure water 50m L m, ultrasonic treating in water bath for 20min, homogenizing in high pressure homogenizer at 200bar for 30s, adding 150bar every 30s until 800bar, and finally continuing at 800bar for 20 cycles.

(3) The probe ultrasonic method comprises weighing Saviae Miltiorrhizae radix extract 0.1g and rhizoma Ligustici Chuanxiong extract 0.08g, adding pure water 50m L, performing probe ultrasonic under 1200W power for 2s, and repeating the operation for 2s at interval for 3 min.

(4) High-pressure homogenization after precipitation of the anti-solvent: the suspension was prepared according to method "(1)" and homogenized in a high-pressure homogenizer at 300bar for 10 cycles, 500bar for 15 cycles and 800bar for 20 cycles.

(5) Probe ultrasonic method after anti-solvent precipitation: prepare the suspension according to the method (1), and then perform ultrasonic probe under 1200W power for 2s, 2s pause and 3 min.

(6) An anti-solvent precipitation method under water bath ultrasound comprises adding 0.1g Saviae Miltiorrhizae radix extract and 0.08g rhizoma Ligustici Chuanxiong extract into 5m L90% ethanol, performing ultrasound for 15min, centrifuging at 5000rpm for 10 min.50m L pure water, adding into 250m L three-neck flask, placing into 300W ultrasound water bath, rapidly adding the above centrifugated supernatant under ultrasound and 1000rpm electric stirring, dissolving the centrifugated precipitate with 1m L pure water, adding into the flask together, continuing performing ultrasound stirring for 30min, connecting with a pressure reducing device, and removing ethanol under reduced pressure (pressure less than-0.09 Mpa) and ultrasound for 30 min.

(7) Precipitating the anti-solvent under the water bath ultrasound and then performing probe ultrasound: prepare the suspension according to the formula (6), and then perform ultrasonic treatment under 800W power for 2s with 2s interval for 3 min.

The particle size and distribution of the nanosuspension prepared by the preparation method of 7 above were examined. Measuring with a particle size analyzer to obtain D₅₀PDI, number percentage of particles smaller than 1 μm, etc., and the results are shown in table 2.

Table 2 particle size and distribution of nanosuspensions from different preparation methods (n ═ 3)

Analysis and conclusion: table 2 shows that the probe ultrasound method cannot directly prepare the extract into a nanosuspension, and only less than 3% of the particles have a particle size below 1 μm; the grain diameter sequence obtained by the other methods is as follows from large to small: high-pressure homogenization method, anti-solvent precipitation method under water bath ultrasound, probe ultrasound method after anti-solvent precipitation, high-pressure homogenization method after anti-solvent precipitation method, anti-solvent precipitation method under water bath ultrasound and probe ultrasound. The ultrasonic method of the anti-solvent precipitation under the water bath ultrasonic is carried out, then the probe ultrasonic is carried out, the high-pressure homogenization method after the anti-solvent precipitation and the probe ultrasonic method after the anti-solvent precipitation have more than 90 percent of particles in the nanometer grade. PDI is maximum and exceeds 0.3 by an anti-solvent precipitation method under water bath ultrasound; the other methods are all between 0.1 and 0.3. Therefore, the particle size of the nanometer suspension prepared by the anti-solvent precipitation under the water bath ultrasound and the probe ultrasound is smaller, more than 90 percent of particles are below 1 mu m, and the particle size distribution is uniform.

The processes of the antisolvent method of the present invention all involve the addition of an organic phase to an aqueous phase. Unlike the monomer insoluble medicine which can be completely dissolved in some organic solvent, the traditional Chinese medicine extract usually contains both water-soluble components and insoluble components. The preliminary experiment shows that 90% v/v ethanol and absolute ethanol can well dissolve insoluble components in the salvia miltiorrhiza and ligusticum wallichii extracts, but the dissolving amount of water-soluble components in the 90% v/v ethanol is more than that of the absolute ethanol, so that the 90% v/v ethanol is selected as an organic phase in the anti-solvent process. However, 90% v/v ethanol can not completely dissolve the extracts of the salvia miltiorrhiza and the ligusticum wallichii, and obvious precipitates appear after centrifugation. In view of the fact that the precipitate is not determined to be an ineffective component, in order to ensure the curative effect of the preparation, in the research, after the supernatant of the centrifuged organic phase is added into the water phase, the precipitate is completely dissolved by pure water and then added together, and an anti-solvent precipitation method suitable for the ligusticum wallichii extract and the salvia miltiorrhiza extract is created.

The results of examining the total yield and the distribution percentage in the nanometer scale (particle size 10-1000 nm) of the insoluble component in the suspension prepared by the preparation method of 7 above are shown in tables 3-1 and 3-2.

1) And (3) measuring the total yield, namely shaking a newly prepared sample uniformly, immediately sampling 0.5m L, adding a proper amount of methanol, performing ultrasonic dissolution for 5min, fixing the volume to 1m L, filtering by using a 0.22 mu m microporous membrane, measuring the content of each component by using HP L C, and comparing the content with the dosage to calculate the total yield.

2) The mass percentage of the insoluble components in the suspension at the nanometer level is measured by taking 10m L nanometer suspension, pumping and filtering the suspension by using a 1000nm track etching film, filtering the filtrate by using a 100nm track etching film, taking a 100nm filter membrane, adding 5m L80% methanol, carrying out ultrasonic treatment for 30min, dissolving and precipitating, repeating for 1 time, combining 2 ultrasonic solutions, filtering for 0.22 mu m, measuring the contents of senkyunolide A, ligustilide, butenyl phthalide, cryptotanshinone, tanshinone I and tanshinone II A by using HP L C, comparing the contents with the total mass of the components in the suspension, and calculating the percentage of the components in the nanometer level (100-1000 nm) range.

TABLE 3-1 Total yield of poorly soluble component and nanoparticle yield (n ═ 3)/% of suspensions from different preparation methods

TABLE 3-2 Total yield of poorly soluble component and nanoparticle yield (n ═ 3)/% of suspensions from different preparation methods

Analysis and conclusion: as can be seen from tables 3-1 and 3-2:

1) the total yield of each component in the suspensions prepared by the high pressure homogenization method and the high pressure homogenization method after the precipitation of the anti-solvent is low in terms of the total yield.

2) In terms of distribution of each component, in the suspension prepared by the anti-solvent precipitation method, the anti-solvent precipitation method under water bath ultrasound and the probe ultrasound method after the anti-solvent precipitation under water bath ultrasound, the yield of 6 insoluble components of senkyunolide A, ligustilide, butenyl phthalide, cryptotanshinone, tanshinone I and tanshinone IIA is higher in the nano-scale range.

3) The particle size, the total yield and the distribution rate of 100-1000nm are comprehensively considered, and the optimal method is a probe ultrasonic method after the anti-solvent is precipitated under the water bath ultrasonic, and then the probe ultrasonic method after the anti-solvent is precipitated and an anti-solvent precipitation method under the water bath ultrasonic.

2. Screening and optimizing process conditions for anti-solvent precipitation process

Precipitating with antisolvent under optimal water bath ultrasound, preparing perhexiline nanometer suspension by probe ultrasound method, and further selecting total concentration (X) of extract in ethanol solution₁) Volume ratio of ethanol to aqueous phase (X)₂) Water bath temperature (X)₃) For inspectionFactors, the Box-Behnken design is used for optimizing the anti-solvent step in the preparation process, and the process conditions are optimized.

Total concentration of extract in ethanol solution (X)₁) Volume ratio of ethanol to aqueous phase (X)₂) Water bath temperature (X)₃) For the factor, the factor-level for the Box-Behnken design is shown in Table 4 and the experimental design is shown in Table 5.

The inventor finds in research that the concentration of a drug affects the stability of a system, the concentration is increased to increase the relative saturation concentration, the more nanocrystal nuclei are formed, but the mutual collision contact between the crystal nuclei is increased to easily cause aggregation, the larger the volume of ethanol is, the larger the saturated solubility of the drug in an ethanol-water mixed solvent is, the less the supersaturated concentration is, the more adverse to the formation of nanocrystal nuclei, the crystallization temperature when an organic solvent is injected into an anti-solvent is reduced, the solubility of the drug in the anti-solvent is reduced to increase the relative supersaturation concentration, the formation of the crystal nuclei is facilitated, the lower the temperature is, the slower the growth of the crystal nuclei is, the cross-linking effect of the three factors can be caused, and a single-factor test shows that the ultrasonic intensity is affected by the volume of a liquid in an ultrasonic pool, so that the volume of a fixed anti-solvent is 50m L, the volume of 90% ethanol is 1-5 m L, and the concentration of the drug in an organic phase is 0.036-0.252 g. L g^-1(ii) a The low temperature is favorable for the nucleation of the nanocrystalline, and the ligusticum wallichii extract contains components such as ligustilide which are likely to volatilize at the high temperature, so the temperature range is set to be 0-40 ℃, and the factor and the level of the Box-Behnken design in the invention are determined.

TABLE 4 factor level table for Box-Behnken design

TABLE 5Box-Behnken Experimental arrangement

The optimization indexes are as follows:

by particle size (Y)₁)、PDI(Y₂) And a fine particle percentage of 1 μm or less (Y)₃) And a normalized value (Y) of the percentage distribution of the poorly soluble component at 100 to 1000nm₄) Is used as an index. The measurement method of each index is as follows:

particle size (Y)₁)、PDI(Y₂) And a percentage of particles (Y) of 1 μm or less₃) The prepared sample is immediately used for measuring the particle diameter by using a nano laser particle analyzer to directly obtain D₅₀PDI and the percentage of particles below 1 μm.

Normalized value (Y) of distribution percentage of 6 insoluble components in 100-1000nm₄) And measuring the distribution percentage of senkyunolide A, ligustilide, butenyl phthalide, cryptotanshinone, tanshinone I and tanshinone IIA in the suspension in the range of 100-1000nm, and performing normalization treatment according to the percentage of node connectivity of each component in the network pharmacology result (see table 6).

TABLE 6 weight percent settings for each component of the normalization process

Normalized value (Y) of distribution percentage of each component of 100 to 1000nm₄) The formula of (1) is as follows:

Y₄cnidium officinale lactone A distribution rate × 0.07.07 + ligustilide distribution rate × 0.22.22 + butenyl phthalide distribution rate × 0.26.26 + cryptotanshinone distribution rate × 0.13.13 + tanshinone I distribution rate × 0.06.06 + tanshinone IIA distribution rate × 0.26.26

The preparation method comprises preparing 90% ethanol solution of Saviae Miltiorrhizae radix and rhizoma Ligustici Chuanxiong extract (fixed Saviae Miltiorrhizae radix extract: rhizoma Ligustici Chuanxiong extract: 10:8) with certain volume and certain concentration according to Table 5, performing ultrasonic treatment for 15min, centrifuging at 5000rpm for 10min, adding 50m L pure water into 250m L three-neck flask, placing in ultrasonic water bath at certain temperature, stirring at 1000rpm, rapidly adding centrifugated supernatant under 300W ultrasonic treatment, dissolving precipitate with 1m L pure water, adding together, continuing ultrasonic stirring for 30min, connecting decompression device, removing ethanol under reduced pressure (pressure less than-0.09 Mpa) and water bath ultrasonic treatment for 30min, and performing ultrasonic treatment on the obtained suspension with probe at certain power for a certain time.

Data processing and analysis:

using SPSS software according to Y ═ A₀+A₁X₁+A₂X₂+A₃X₃+A₄X₁X₂+A₅X₁X₃+A₆X₂X₃+A₇X₁ ²+A₈X₂ ²+A₉X₃ ²Performing quadratic multiple regression fitting on the form of the expression, and deleting the items with larger P values one by one until the regression equation is highly significant. According to the test schedule table of table 5, except 2 factors to be examined, the other variables are set as median values (i.e., values of 0 level), and the theoretical Y value is calculated according to the binomial equation. Respectively drawing three-dimensional effect surface and two-dimensional contour map of each index and 2 independent variables with obvious influence by Origin 6.0 software, and inspecting total concentration (X) of the extract in ethanol solution₁) Volume ratio of ethanol to aqueous phase (X)₂) And temperature of the water bath (X)₃)。

Box-Behnken design optimization result

17 parts perhexiline nanosuspension were prepared as in Table 5. The distribution rate results of the insoluble components in each sample in the range of 100-1000nm are shown in Table 7, and the optimization evaluation index results are shown in Table 8.

TABLE 7 distribution ratio/% of nanoparticles of 6 poorly soluble Components in each sample designed by Box-Behnken

TABLE 8 evaluation results of Box-Behnken design for each sample

As a result: the three-dimensional effect surface and the two-dimensional isometric view show that: the lower drug concentration, the proportion of the organic phase to the aqueous phase and the temperature are beneficial to reducing the particle size and PDI of the nano suspension; compared withThe low drug concentration and the ratio of the organic phase to the aqueous phase are favorable for the formation of particles with the particle size of less than 1 mu m, and the temperature is 10-25 ℃ which is favorable for the formation of particles with the particle size of less than 1 mu m; the normalized value of the distribution rate of 100-1000nm is higher in higher drug concentration, lower proportion of organic phase and aqueous phase and temperature. The technological condition of the anti-solvent process is the total concentration (X) of the extract in the ethanol solution₁) Volume ratio of ethanol to aqueous phase (X)₂) And temperature of the water bath (X)₃) The screening results are shown in Table 9.

TABLE 9 key factors of the antisolvent procedure

3. Screening and optimizing ultrasonic process conditions

Preparing the perhexiline primary suspension according to the anti-solvent precipitation process designed and optimized by Box-Behnken, and carrying out probe ultrasound on the obtained primary suspension, wherein the process condition of ultrasound is optimized.

(1) Optimization of ultrasonic power

Setting the ultrasonic power of the probe at 800W, 1200W, 1600W and 2000W, fixing the ultrasonic time for 3min, performing probe ultrasonic treatment on the primary suspension, and measuring the particle size and the percentage of insoluble components in micron and nanometer.

Optimizing the ultrasonic power of the probe: the particle size, PDI and the percentage test result of particles with the particle size less than 1 μm of the suspension prepared by different ultrasonic powers are shown in a table 10, and the distribution rate result of 100-1000nm is shown in a table 11.

Table 10 particle size, distribution and percentage of particles smaller than 1 μm (n ═ 3) for suspensions prepared at different probe ultrasonic powers

Note: the SPSS software single-factor variance analysis shows that the same letter represents that the same index of different ultrasonic powers has no significant difference, and different letters represent that the same index has significant difference.

TABLE 11 distribution ratio (n-3)/% of 6 kinds of poorly soluble components in suspensions prepared with different probe ultrasonic powers

Note: and (3) SPSS software single-factor variance analysis, wherein the same letter indicates that the distribution rate of the same component under different powers has no significant difference, and different letters indicate that the distribution rate has significant difference.

From tables 10 and 11, it can be seen that in the range of 800-1600W, when the ultrasonic power of the probe is increased to 1600W, the particle size is significantly reduced, the number of particles below 1 μm is significantly increased, but when the ultrasonic power is continuously increased to 2000W, the particle size is not significantly changed; the PDI is minimally affected by the probe ultrasound power. The distribution ratio change of each indissolvable component in the range of 100-1000nm is similar to the particle size. By integrating the above results and reducing the loss of the instrument, the ultrasonic power range of the probe can be determined to be 800-.

(2) Optimization of ultrasound time

And setting the probe ultrasonic time to be 3min, 6min and 9min respectively according to the determined optimal probe ultrasonic power, carrying out probe ultrasonic treatment on the primary suspension, and carrying out evaluation in the same way.

The particle size, PDI and particle percentage test results of particles with the particle size less than 1 μm in different probe ultrasonic time are shown in Table 12, and the distribution rate results of 100-1000nm are shown in Table 13.

Table 12 particle size, distribution and percent of particles of nanosuspensions prepared at different probe sonication times (n ═ 3)

Note: and in SPSS software single-factor variance analysis, the same letter indicates that the result of the same index at different ultrasonic time has no significant difference, and the different letters indicate that the result has significant difference.

TABLE 13 distribution ratio (n-3)/% of each poorly soluble component in nanosuspension prepared at different probe sonication times

Note: the SPSS software single-factor variance analysis shows that the distribution rate of the same component in different ultrasonic time has no significant difference by the same letter and has significant difference by different letters.

As can be seen from tables 12 and 13, as the probe ultrasonic time increases, the particle size decreases, but when the time increases to 9min, there is no significant difference compared to 6 min; PDI, the percentage of particles below 1 μm is not affected by the probe's ultrasound time. The total distribution rate of 100-1000nm is increased along with the increase of the ultrasonic time. And integrating the results and reducing the loss of the instrument, and determining the ultrasonic time of the probe to be more than 3min, preferably 5-7 min.

Second, screening of the stabilizer

1. Screening of stabilizer varieties

Poloxamer188 (hereinafter abbreviated as Poloxamer 188), polyvinylpyrrolidone-K30 (hereinafter abbreviated as PVP K30), Tween80 (hereinafter abbreviated as Tween 80), sodium dodecyl sulfate (hereinafter abbreviated as SDS), polyvinyl alcohol (hereinafter abbreviated as PVA) and hydroxypropyl methyl cellulose K4M (hereinafter abbreviated as HPMC K4M) are respectively used as alternative stabilizers, all the stabilizers are added into an aqueous phase according to a medicine-auxiliary ratio of 1:1, a probe ultrasonic method is carried out after the precipitation of a antisolvent under water bath ultrasonic to prepare a nano suspension, the prepared sample is stood at room temperature, supernatant fluid of senkyunolide A, ligustilide, butenyl phthalide, cryptotanshinone, tanshinone I and tanshinone IIA is absorbed at 0, 1, 3 and 7d, the content of senkyunolide A, ligustilide, butenyl phthalide, cryptotanshinone, tanshinone I and tanshinone IIA is measured, and the particle size, PDI and potential are measured after the rest samples are shaken uniformly.

The particle size, PDI and Zeta potential results of the nano suspensions prepared by different stabilizers after standing for different times are respectively shown in tables 14-16, and the content change of insoluble components in the upper suspension is shown in table 17.

TABLE 14 particle size D for different stabilizers on standing for different times₅₀Variation (n ═ 3)

Note: SPSS software one-way anova, "+" indicates P <0.05 and "+" indicates P <0.01, compared to time 0.

As can be seen from table 14, HPMC K4M increased particle size very significantly; poloxamer188 and PVP K301 d showed significant particle size increase, PVA showed significant particle size increase at 7d, and Tween80, SDS and HPMC K4M showed no significant change in particle size after standing for 7 d.

TABLE 15 PDI change for different stabilizers on standing for different times (n ═ 3)

Note: SPSS software one-way anova, "-" indicates P <0.05 compared to time 0.

According to the table 15, the nano-suspension prepared from PVP K30, SDS and HPMC K4M has small PDI, which indicates that the particle size is uniform and is obviously superior to Poloxamer188, Tween80 and PVA; after standing for 7d, the particle size distribution of Poloxamer188, PVP K30 showed a significant increase, and the remaining stabilizers were not significantly changed.

TABLE 16 suspension of different stabilizers with potential Zeta variation (n 3)/mV at different times of standing

Note: SPSS software one-way anova, "-" indicates P <0.05 compared to time 0.

According to Table 16, the Zeta potential of the nanosuspension prepared by SDS is the lowest, and the negative charge of the nanoparticles is the most; the Zeta potential of the nanometer suspension prepared by HPMC K4M is highest, and the negative charge of the nanometer particle is least and is close to 0; the rest of the stabilizing agent is between the two. Significant changes of PVA and HPMC K4M appear after 3d, significant changes of Poloxamer188 and PVP K30 appear after 7d, and no significant change of potential occurs after the Tween80 and SDS are placed for 7 d.

TABLE 17 content change of insoluble component by standing different stabilizers for different periods of time (n-3)/mg-50 m L^-1

Note: SPSS software one-way anova, "+" indicates P <0.05 and "+" indicates P <0.01, compared to time 0.

The results in table 17 show that Poloxamer188, PVP K30, PVA, HPMC K4M could not improve the stability of the perhexiline nanocrystalline suspension, and significant content reduction occurred after the senkyunolide a, ligustilide, tanshinone IIA were left to stand for a certain time; the Tween80 and SDS groups have no significant change in content after standing for 7 days.

From the results, different stabilizers have great influence on the particle size, PDI and potential, the particle size and distribution of the nanosuspension prepared from Poloxamer188, PVP K30 and PVA are changed greatly in the standing process, and obvious precipitation visible to naked eyes appears after standing for 24 hours; HPMC K4M has good stability but large particle size; the nano suspension prepared from Tween80 and SDS is still in the range of nano crystals, and the changes of particle size, charge and content of insoluble components are small after the nano suspension is placed for 7 days.

In view of the results in tables 14 to 17, SDS and Tween80 were preferably used as a stabilizer.

2. Screening of stabilizer dosage

SDS and Tween80 are added into the water phase according to the drug-adjuvant ratio of 1:0.25, 1:0.5 and 1:1, the results of the particle size, PDI and Zeta potential of 0, 1, 3 and 7d of the nano-suspension prepared by adopting different amounts of SDS or Tween80 are shown in tables 18-20, and the content change of the insoluble component in the supernatant is shown in table 21.

TABLE 18 particle size D of nanosuspensions prepared with different amounts of stabilizer left for different periods of time₅₀Variation (n ═ 3)

Note: SPSS software one-way anova, "-" indicates P <0.05 compared to time 0.

As can be seen from table 18, the particle size of the nanosuspensions prepared by SDS with different drug-adjuvant ratios is significantly lower than Tween 80; in the range of the drug-adjuvant ratio of 1:0.25-1:1, the dosage of the adjuvant has no significant influence on the particle size of a newly prepared nano suspension of the same stabilizer; however, when the dose-adjuvant ratio of Tween80 is 1:0.25 and 1:0.5, the particle size of the sample is remarkably increased after standing for 3d and 7d respectively.

TABLE 19 distribution of different amounts of stabilizer on standing (n. 3)

Note: SPSS software one-way anova, "-" indicates P <0.05 compared to time 0.

From table 19, it can be seen that, in the range of the drug-adjuvant ratio of 1:0.25-1:1, the dosage of the adjuvant has no significant influence on PDI of a freshly prepared nanosuspension of the same stabilizer; the drug-adjuvant ratio of the Tween80 is 1:0.25, and 1:0.5, and the PDI is remarkably increased after standing for 3d and 7d respectively; the drug-adjuvant ratio of SDS is 1:0.25, 1:0.5, and the PDI is obviously increased after standing for 1 d.

TABLE 20 Zeta potential values for different periods of time (n. 3) for samples prepared with different amounts of stabilizer

Note: SPSS software one-way anova, "×" indicates P <0.01 compared to time 0.

From table 20, it can be seen that in the range of the drug-adjuvant ratio of 1:0.25-1:1, the amount of the adjuvant has no significant influence on the potential of the freshly prepared nanosuspension of the same stabilizer, and after standing for 7 days, the zeta potential of 2 samples with the drug-adjuvant ratio of 1:0.25 is significantly increased.

TABLE 21 content variation of insoluble component (n-3)/mg-50 m L for samples prepared with different amounts of stabilizer^-1

Note: SPSS software one-way anova, "+" indicates P <0.05 and "+" indicates P <0.01, compared to time 0.

As can be seen from table 21, the amount of the stabilizer has a certain effect on the stability of the nanosuspension, and the samples with the drug-adjuvant ratio of 1:0.25 and 1:0.5 all showed visible precipitation after standing for 7 days. After 7 days, when the auxiliary ratio of 2 auxiliary materials is 1:0.25 and 1:0.5, the content reduction rate of cryptotanshinone is 58.33-89.47%; the content of tanshinone I is reduced by 23.08-44.44 percent, and the content of tanshinone IIA is reduced by 25.00-337.50 percent. When the medicine-auxiliary ratio is 1:1, the retention rate of senkyunolide A, ligustilide and butenyl phthalide in the suspension with the stabilizing agent Tween80 is higher than that of SDS, but the retention rate of cryptotanshinone, tanshinone I and tanshinone IIA is lower than that of SDS.

By querying the database of commonly used excipients in the drug review center of the State drug administration, the addition dose of SDS (1: 1 for drug-adjuvant) in the optimal results of the study already exceeded the maximum allowable dose (0.25%), but Tween80 in the 1:1 for drug-adjuvant did not exceed the maximum allowable dose, and the results were better than the other results of SDS.

Finally determining the drug-adjuvant ratio to be 1:0.25-1:1 by integrating particle size, PDI, potential, and standing content results and safety, wherein the drug-adjuvant ratio is 1:1 by adopting Tween80 as a stabilizer; SDS is used as a stabilizer, and the drug-adjuvant ratio is 1:0.25-1:1, preferably 1: 1.

Particularly, in the research process, the Tween80 is used as a stabilizer, and compared with the nano suspension without the Tween80, the senkyunolide A, the ligustilide, the butenyl phthalide, the cryptotanshinone, the tanshinone I and the tanshinone II A in the nano suspension have the advantages that the distribution is reduced by 100-400 nm, and the distribution below 100nm is increased. The Tween80 has a certain solubilization effect, and the percent of the Tween80 below 100nm is obviously increased compared with that of the Tween80 which is not added in the process of preparing the nano suspension. Therefore, Tween80 is most preferably selected as the stabilizer of the present invention.

Preparation of nano freeze-dried powder

Preparing nanometer suspension according to the optimized result, respectively adding 4%, 8% and 12% of freeze-drying protective agent (alternative varieties: mannitol, lactose, glucose, dextran and maltose), stirring for dissolving, then subpackaging in penicillin bottles (2m L/bottle), pre-freezing at 20 ℃ for 2h, then transferring to an ultra-low temperature refrigerator at-80 ℃ for pre-freezing for 24h, then drying at 15 ℃ for 24h under the vacuum degree of below 15mTorr, taking out and immediately sealing, wherein the appearance diagram of each freeze-drying protective agent is shown in figure 1, and the particle size of the freeze-dried sample is shown in tables 22-24 after re-dispersion.

TABLE 22 particle size of redispersed samples of different lyoprotectants

Note: SPSS software single-factor analysis of variance shows that the same letter indicates that no significant difference exists when the concentration of each cryoprotectant is 4%, and different letters indicate that significant differences exist; the same lyoprotectant indicates P <0.05, "×" indicates P <0.01, compared to a concentration of 4%.

Analysis and conclusion:

FIG. 1 shows: maltose and mannitol can be well dissolved in the suspension, and a freeze-dried product has a smooth surface and loose texture and can be well re-dispersed; the dextran has low solubility in the nano suspension and is turbid, the surface of a freeze-dried product with the concentration of 4% shrinks, and when the dosage is increased to 8% and 12%, the shrinking phenomenon is relieved, but the dextran still becomes turbid after redispersion; lactose and glucose can be well dissolved in the suspension, the surface of a freeze-dried product with the concentration of 4% is obviously shrunk and collapsed, after the dosage is increased to 8% and 12%, the shrinking phenomenon of lactose is relieved but the shrinking phenomenon still exists, and the tendency of bottle spraying is realized instead of the condition that the shrinking phenomenon of glucose is not relieved.

As can be seen from table 22, when the addition amount is 4%, the particle size of the lyophilized powder using dextran as the lyoprotectant is the largest after redispersion, and there is no significant difference between mannitol, maltose, lactose and glucose; when the addition amount is increased to 8% and 12%, the particle size of the dried powder taking maltose, mannitol and glucose as the freeze-drying protective agent after redispersion is not significantly different from that of 4%, the particle size of the freeze-dried powder taking lactose as the protective agent after redispersion is significantly different and increased, and the particle size of the freeze-dried powder taking dextran as the freeze-drying protective agent after dispersion is significantly different and increased.

TABLE 23 post-redispersion distribution values for different lyoprotectants

Note: SPSS software single-factor analysis of variance shows that the same letter indicates that no significant difference exists when the concentration of each cryoprotectant is 4%, and different letters indicate that significant differences exist; the same lyoprotectant, "-" indicates P <0.05 compared to 4% concentration.

From the distribution results table 23, when the addition amount is 4%, the PDI is smaller after the freeze-dried powder of 2 kinds of freeze-drying protective agents of maltose and lactose is re-dispersed, and the contents of mannitol, glucose and dextran are larger; when the addition amount is increased to 8%, PDI (Poly-propylene-diene monomer) of the freeze-dried powder which takes maltose and lactose as freeze-drying protective agents is not significantly changed after being redispersed, PDI of mannitol and glucose is significantly reduced, and PDI of dextran is significantly increased; when the addition amount is increased to 12%, compared with 4%, PDI of the maltose and glucose freeze-dried powder after redispersion is not significantly changed, PDI of the dextran and lactose freeze-dried powder after redispersion is significantly increased, and PDI of the mannitol freeze-dried powder after redispersion is significantly reduced.

TABLE 24 Zeta potentials after redispersion of different lyoprotectants samples

Note: SPSS software single-factor analysis of variance shows that the same letter indicates that no significant difference exists when the concentration of each cryoprotectant is 4%, and different letters indicate that significant differences exist; "-" indicates that P <0.05 was compared to the same lyoprotectant at a concentration of 4%.

From table 24, it can be seen that when the dosage is 4%, the charge of the lyophilized powder using dextran as the lyoprotectant is less after redispersion, and the charge of mannitol, lactose and glucose is more; when the addition amount is increased to 8%, the Zeta potential of the freeze-dried powder taking maltose, mannitol, dextran and glucose as the freeze-drying protective agent after redispersion is not significantly changed, and the charges of the freeze-dried powder taking lactose as the freeze-drying protective agent after redispersion are significantly reduced; when the addition amount is increased to 12%, compared with 4%, the Zeta potential of the freeze-dried powder taking maltose, dextran and glucose as the freeze-drying protective agent after redispersion is not significantly changed, and the charge of the freeze-dried powder taking mannitol and lactose as the freeze-drying protective agent after redispersion is reduced.

So the final optimization conditions are as follows: maltose, mannitol, glucose and lactose are taken as freeze-drying protective agents, and the dosage is 4-12%; mannitol is preferably used as the lyoprotectant, preferably in an amount of 4-12%.

Fourth, evaluation of Effect

Sample preparation:

(1) the crude suspension of the raw materials is prepared by weighing 0.14g of Salvia miltiorrhiza extract and 0.11g of Ligusticum chuanxiong extract, adding 50m L pure water, and stirring.

(2) The nanometer suspension is prepared by adding 0.14g Saviae Miltiorrhizae radix extract and 0.11g rhizoma Ligustici Chuanxiong extract into 1.5m L90% ethanol, performing ultrasonic treatment for 15min, centrifuging at 5000rpm for 10min, dispersing 0.25g Tween80 into 50m L pure water, adding into 250m L three-neck flask, placing in ultrasonic water bath at 12 deg.C and 300W power, rapidly adding the above centrifugated supernatant under ultrasonic and 1000rpm electric stirring, dissolving the centrifugal precipitate with 1m L pure water, adding, continuing ultrasonic stirring for 30min, connecting with pressure reducer, reducing pressure (pressure less than-0.09) and removing ethanol under ultrasonic condition for 30min, and performing ultrasonic treatment with 1600W probe for 6 min.

(3) The nanometer lyophilized powder is prepared by collecting Saviae Miltiorrhizae radix extract 0.14g, rhizoma Ligustici Chuanxiong extract 0.11g, adding 1.5m 3690% ethanol, performing ultrasonic treatment for 15min, centrifuging at 5000rpm for 10min, dispersing Tween80 0.25g in 50m L pure water, adding into 250m L three-neck flask, placing in ultrasonic water bath with power of 12 deg.C and 300W, ultrasonically stirring at 1000rpm, rapidly adding the above centrifugated supernatant, dissolving the centrifugal precipitate with 1m L pure water, adding, continuously performing ultrasonic stirring for 30min, connecting with decompressor, decompressing (pressure less than-0.09) and removing ethanol under ultrasonic condition for 30min, adding mannitol 8% into 1600W probe ultrasonic for 6min, stirring for dissolving, packaging in penicillin bottle (2m L/bottle), prefreezing at 20 deg.C for 2h, transferring into-80 deg.C refrigerator, prefreezing for 24h, freeze drying at room temperature of 15mTorr pressure, taking out, and immediately sealing.

1. Particle size distribution and Zeta potential: samples (1) and (3) were re-dispersed in suspension, and sample (2) was measured for particle size and Zeta potential using a laser particle size distribution analyzer and a Zeta potential analyzer.

The particle size distribution and Zeta potential measurement results of the coarse suspension, the nano suspension and the nano freeze-dried powder redispersed suspension are shown in table 25.

TABLE 25 particle size, distribution and Zeta potential of the different suspensions (n ═ 3)

Note: and (3) SPSS software single-factor analysis of variance, wherein the same letters indicate that the same index has no significant difference, and different letters indicate that the index has significant difference.

The results in table 25 show that the nanosuspension technology can reduce the particle size of the perhexiline drug substance to the nanometer level, and simultaneously significantly reduce the PDI and Zeta potential, and the nanosuspension has a very significant difference (P <0.01) compared with the coarse suspensoid; compared with the suspension before freeze-drying, the particle size, PDI and Zeta potential of the nano freeze-dried powder have no significant change, which shows that the particle size and the charge property of the nano particles are not influenced by freeze-drying.

2. The method comprises the following specific operations of taking 10m L coarse suspension, nano suspension and nano freeze-dried powder redispersed liquid, respectively carrying out suction filtration by using a 1-micron track etching membrane, carrying out ultrasonic treatment on a filter membrane by adding 5m L80% methanol for 30min, dissolving and precipitating, repeating for 1 time, combining 2 times of methanol solution, filtering by 0.22 micron, measuring the content of 14 index components by using HP L C to obtain the composition and the content of particles with the particle size of more than 1 micron, carrying out suction filtration on filtrate by using a 400-nm track etching membrane, obtaining the composition and the content of the particles with the particle size of 400 nm-1 micron, carrying out suction filtration on the filtrate by using a 100-nm track etching membrane, obtaining the composition and the content of the particles with the particle size of 100 nm-400 nm, measuring the final filtrate, obtaining the composition and the content of the particles with the particle size of less than 100nm and the components in a molecular state, and measuring the content of the coarse suspension, the nano suspension and the freeze-dried powder after the classification filtration, wherein the measurement results are shown in Table 26.

Table 26 shows that salvianic acid, protocatechuic aldehyde, ligustrazine, ferulic acid, senkyunolide I, senkyunolide H, rosmarinic acid, salvianolic acid B in the suspension redispersed by the crude suspension, the nanosuspension, and the nano lyophilized powder have relatively high solubility in water, and are uniformly distributed in the filtrate filtered at 100 nm; the crude suspension contains ligustilide A (23.11 + -0.58)% greater than 1 μm, (70.62 + -11.10)% less than 100nm, and ligustilide, butenyl phthalide, cryptotanshinone, tanshinone I and tanshinone IIA greater than 1 μm are more than 80%. The nano suspension technology can obviously reduce the mass ratio of senkyunolide A, ligustilide, butenyl phthalide, cryptotanshinone, tanshinone I and tanshinone IIA with the grain diameter of more than 1 mu m: in the nanosuspension, 90% or more of the 6 components are distributed below 400 nm. Compared with the suspension before freeze-drying, the content measurement result of 14 components such as danshensu and the like in the suspension after the freeze-dried powder is re-dispersed is not obviously changed after graded filtration, which shows that the particle size distribution of each component is not influenced in the freeze-drying process.

Table 26 content of 14 ingredients in different particle size ranges after fractional filtration of different suspensions (n-3)/mg 50m L^-1

Note: SPSS software single-factor variance analysis, wherein the same letters indicate that the same index has no significant difference, and different letters indicate that the index has significant difference; "-" was not detected.

3. Measuring apparent solubility by taking excessive raw materials (10: 8 physical mixture of Saviae Miltiorrhizae radix extract and rhizoma Ligustici Chuanxiong extract) and lyophilized powder, adding pure water 3m L, vortex mixing for 2min at 37 deg.C, oscillating at 100rpm, taking out at 48 and 72 hr, and measuring 15000r min^-1Centrifuging for 15min, collecting supernatant 100 μ L L C, and determining content of each component, apparent solubility of each component is shown in Table 27.

Table 27 apparent solubility (n ═ 3)/μ g · m L of each component in the perhexiline bulk drug and the nano freeze-dried powder^-1

Note: no detection; compared with the bulk drug, "+" indicates P <0.05, and "+" indicates P < 0.01.

4. In vitro dissolution, 1 Guanxinning tablet, raw material medicines (physical mixture of salvia extract and ligusticum wallichii extract 10:8) and a proper amount of freeze-dried powder (each sample amount is calculated by salvianolic acid B and is equivalent to 16.53mg) are taken and put into 900m L release media (simulated gastric juice and simulated intestinal juice respectively), the mixture is stirred at 37 ℃ and 50rpm, samples are taken for 2m L and 100nm microporous filter membrane filtration at 5, 10, 15, 30, 45, 60, 90 and 120min respectively, and the contents of danshensu, protocatechuic aldehyde, ligustrazine, ferulic acid, senkyunolide I, senkyunolide H, rosmarinic acid, salvianolic acid B, senkyunolide A, ligustilide, butenyl phthalide, cryptotanshinone I and tanshinone II in the filtrate are measured by HP L C.A release curve of the Guanxinning raw material medicines and the freeze-dried powder of the nano suspension is shown in figure 2.

FIG. 2 shows: the cumulative release rates of the water-soluble components in the raw material medicine and the nano freeze-dried powder in simulated gastric juice and simulated intestinal juice are higher than 90 percent. The perhexiline tablet hardly disintegrates and releases in simulated gastric fluid in the first 15min, and the accumulative release rate of each water-soluble component in 240min is lower and does not exceed 40%; the simulated intestinal juice is disintegrated faster than the simulated gastric juice, but the cumulative release rate of each water-soluble component in 360min is still lower than that of the bulk drug and the nano freeze-dried powder and is not more than 60%. Insoluble components of senkyunolide A, ligustilide and butenyl phthalide water in the raw material drugs have low accumulative release rate in simulated gastric juice and simulated intestinal juice, and the accumulative release rate of cryptotanshinone, tanshinone I and tanshinone IIA is zero; the cumulative release rates of the senkyunolide A, the ligustilide and the butenyl phthalide water-insoluble components in the freeze-dried powder in 120min in simulated gastric fluid are respectively 1.77, 2.26 and 1.82 times of those of the raw material medicines, and the cumulative release rates of the cryptotanshinone, the tanshinone I and the tanshinone IIA are respectively (34.55 +/-3.36)%, (38.65 +/-0.83)%, and (41.08 +/-2.39)%; the cumulative release rates of the senkyunolide A, the ligustilide and the butenyl phthalide water-insoluble components in the freeze-dried powder in 360min are respectively 2.00, 4.18 and 2.62 times of the raw material medicines in simulated intestinal fluid, and the cumulative release rates of cryptotanshinone, tanshinone I and tanshinone IIA are respectively (72.39 +/-8.29)%, (61.68 +/-6.34)%, and (75.43 +/-2.58)%; the GUANXINNING tablet does not contain water-insoluble components such as senkyunolide A, and is not detected in simulated gastrointestinal fluid.

The inventor also determines the stability of the nano freeze-dried powder, and finds that the particle size, PDI, potential and the content of each component do not have significant changes after the nano freeze-dried powder is placed at 4 ℃ for 6 months, and the storage stability is good.

In conclusion, the nano suspension freeze-dried preparation simultaneously contains the water-soluble active ingredients and the indissolvable active ingredients of the salvia miltiorrhiza and the ligusticum wallichii, the salvia miltiorrhiza extract and the ligusticum wallichii extract are skillfully prepared into a nano suspension system and then freeze-dried, the prepared preparation can still keep the properties of particle size, distribution, potential and the like compared with the original liquid after being redispersed by water, particularly, the apparent solubility of the indissolvable ingredients is obviously improved, the preparation can be effectively dissolved out in a simulated gastric juice test, and a new technical thought is provided for clinical application of the ligusticum wallichii and the salvia miltiorrhiza.

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Improved nano suspension freeze-dried preparation based on perhexiline and preparation method thereof

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