CN107080748B - Ligustrazine Ferulic Acid Solid Lipid Nanoparticles and Its Preparation Method and Application - Google Patents

Abstract

The invention discloses a ligustrazine ferulic acid solid lipid nanoparticle and its preparation method and application. ‑2.0, glyceryl monostearate 200‑500, poloxamer 188 300‑500, sodium stearate 5‑20, purified water 10‑30; in Ligustrazine Ferulic Acid Solid Lipid Nanoparticles Tetramethylpyrazine and ferulic acid have a good synergistic effect, high physical stability, controlled drug release, avoid drug degradation or leakage, good targeting, safety and non-toxicity, etc., and can be used in the treatment of ischemic Clinical medicine for cerebrovascular disease.

Description

Ligustrazine Ferulic Acid Solid Lipid Nanoparticles and Its Preparation Method and Application

technical field

The invention belongs to the technical field of medicine, and relates to a ligustrazine ferulic acid solid lipid nanoparticle and a preparation method and application thereof.

Background technique

Ligustrazine is mainly used clinically for the treatment of ischemic cerebrovascular diseases, and has achieved good curative effect; however, Ligustrazine has a fast metabolism and a short half-life. In order to maintain an effective therapeutic concentration of the drug, frequent administration is required during clinical application. Defects such as easy to cause accumulation and poisoning limit its application; ferulic acid has pharmacological activities such as anti-platelet aggregation, thrombosis, and myocardial protection.

Some pharmacological effects of ligustrazine and ferulic acid are consistent, and the research results show that the combination of the two has obvious synergistic effect. An anti-platelet aggregation compound ferulic acid ligustrazine (FATM) was extracted and isolated, and its structure was confirmed and chemically synthesized; subsequent studies have proved that FATM has anti-thrombotic, anti-platelet aggregation and protection against myocardial ischemia-reperfusion injury in rats. The effects in all aspects are better than those of a single component, and the toxic and side effects are reduced. However, the current research on FATM mainly focuses on the pharmacological activity and content determination, and the research on its preparation is less.

Solid lipid nanoparticle (SLN) is a new type of drug delivery system that emerged in the 1990s. Unlike the liposome bilayer structure with phospholipids as the main component, SLN is composed of a variety of lipid materials (such as three Phthaloglycerin, soybean lecithin, etc.) have the advantages of high physical stability, controlled drug release, avoiding drug degradation or leakage, good targeting, and non-toxicity; encapsulating the drug in a physiologically compatible and resistant Compared with traditional pharmaceutical preparations, it has higher bioavailability in the SLN matrix with good acceptance.

Contents of the invention

In order to overcome the above-mentioned defects in the prior art, the object of the present invention is to provide a ferulic acid ligustrazine solid lipid nanoparticle (FATM-SLN).

The object of the present invention is also to provide a preparation method of the above-mentioned ligustrazine ferulic acid solid lipid nanoparticles.

The object of the present invention is also to provide the application of the ligustrazine ferulic acid solid lipid nanoparticles.

The purpose of the present invention is achieved through the following technical solutions:

In the first aspect, ligustrazine ferulic acid solid lipid nanoparticles consist of the following parts by weight:

Preferably, the ligustrazine ferulic acid solid lipid nanoparticles consist of the following parts by weight:

In the second aspect, the preparation method of the above-mentioned ligustrazine ferulic acid solid lipid nanoparticles comprises the following steps:

Weigh ligustrazine ferulic acid, egg yolk lecithin, butyl acetate, and glyceryl monostearate in a 50mL beaker according to the above parts by weight, and dissolve them in a water bath at 65-75°C to form an oil phase; then weigh the above parts by weight Poloxamer 188, sodium stearate, purified water in a 50mL beaker, stirred and dissolved in a water bath at 65-75°C to form a water phase; inject the oil phase into the water phase, and stirring at 4500-5500 rpm for 5 minutes, ultrasonic treatment for 5 minutes with an ultrasonic probe, and quickly placed in an ice-water bath to cool to room temperature.

Preferably, the dissolution temperature of the water bath is 70° C.; the stirring rate is 5000 rpm; and the power ratio of the ultrasonic probe is 30%.

In the third aspect, the application of the above-mentioned ligustrazine ferulic acid solid lipid nanoparticles in the preparation of drugs for treating ischemic cerebrovascular diseases.

The pharmacological effect of each component among the present invention:

Ligustrazine: anti-thrombotic, anti-ischemia-reperfusion injury, protection of cardiovascular and cerebrovascular system, protection of liver, kidney and other pharmacological effects. It also has biological activities such as antitumor, analgesic, and detoxification.

Ferulic acid: anti-atherosclerosis; anti-m platelet aggregation and thrombus; scavenging nitrite, oxygen free radicals, peroxide dummy nitro; anti-bacterial and anti-inflammatory; anti-tumor; anti-mutation; increase immune function, human vitality and mobility etc.

The present invention has the following advantages:

1. Ligustrazine and ferulic acid have a good synergistic effect in Ligustrazine Ferulic Acid Solid Lipid Nanoparticles of the present invention, and the drug effect of Ligustrazine Ferulic Acid Solid Lipid Nanoparticles is obviously better than Ligustrazine or Ferulicum Efficacy of acid when used alone.

2. Ligustrazine ferulic acid solid lipid nanoparticles of the present invention have high physical stability, can control drug release, avoid drug degradation or leakage, have good targeting and are safe and non-toxic, and can be applied to the treatment of deficiencies. Clinical drugs for hemorrhagic cerebrovascular diseases.

3. The present invention selects PC, F68 and sodium stearate as compound emulsifiers, PC is a natural emulsifier, belongs to animal phospholipids, has better biological activity and human body utilization rate; F68 is a nonionic surfactant, which is commonly used A synthetic emulsifier for injection; sodium stearate is an anionic surfactant with excellent emulsifying ability, and when used in combination with nonionic surfactants, the critical micelle concentration is lower and the emulsifying effect is enhanced. The compound emulsifier Effectively reduce particle size and improve stability.

4. The present invention encapsulates ligustrazine and ferulic acid in a physiologically compatible and well-tolerated SLN framework, which has higher bioavailability compared with traditional pharmaceutical preparations, and provides high physical stability, A new Ligustrazine ferulic acid preparation with good curative effect and few side effects laid the foundation.

Description of drawings

Fig. 1 is the ultraviolet scan chromatogram of ligustrazine ferulic acid solid lipid nanoparticle of the present invention, and wherein A is FATM, and B is FATM-SLN, and C is negative sample;

Fig. 2 is the transmission electron micrograph of ligustrazine ferulate solid lipid nanoparticles of the present invention;

Fig. 3 is the particle size distribution figure of ligustrazine ferulate solid lipid nanoparticles of the present invention;

Fig. 4 is the Zeta potential figure of ligustrazine ferulate solid lipid nanoparticles of the present invention;

Fig. 5 is the in vitro release curve of ligustrazine ferulic acid solid lipid nanoparticles.

Detailed ways

The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention.

In the embodiment of the present invention, SPSS16.0 software was used for statistical analysis, and variance analysis was used for comparison between the means of multiple groups of samples, and P<0.05 was regarded as the difference.

Embodiment 1 Preparation of ligustrazine ferulic acid solid lipid nanoparticles of the present invention

1. Instrument

Shear disperser (Germany IKA Group);

Ultrasonic cell pulverizer (Ningbo Xinzhi Biotechnology Co., Ltd.);

TG16 desktop high-speed centrifuge (Changsha Yingtai Instrument Co., Ltd.);

Nano-ZS90 laser scattering particle size analyzer (Malvan, UK);

759S UV-Vis spectrophotometer (Shanghai Jinghua Technology Instrument Co., Ltd.);

ALPHA-BRUKER infrared spectrometer (Germany BRUKER Spectrum Company).

2. Drugs and reagents

Ferulic acid API, ligustrazine API (Shanghai Aladdin Biochemical Technology Co., Ltd., batch number: 20160303, purity: 98.1%);

Ligustrazine ferulic acid (synthesized by the Pharmacy Laboratory of Changsha Medical College, batch number: 20160416, purity: 98.0%);

Methanol, phosphoric acid, egg yolk lecithin (PC), sodium stearate (Sinopharm Chemical Reagent Co., Ltd., batch numbers: 20160223, 20121114, 20151222, 20131209, methanol is chromatographically pure, phosphoric acid is superior grade, and the rest are analytically pure) ;

Butyl acetate (Jiangsu Qiangsheng Functional Chemical Co., Ltd., batch number: 20120910, analytically pure);

Glyceryl monostearate (Shanghai Hengxin Chemical Reagent Co., Ltd., batch number: 20130107, purity: 95.0%);

Poloxamer 188 (F68) (Shenzhen Youpuhui Co., Ltd., batch number: WPWI625C, purity: 98.5%);

Sephadex G50 (Shanghai Ruji Biological Co., Ltd., batch number: 20151112).

3. Method

Weigh 10mg ligustrazine ferulic acid, 400mg egg yolk lecithin, 1.5ml butyl acetate, 300mg glycerol monostearate in a 50mL beaker, dissolve in a 70°C water bath to form an oil phase; then weigh poloxamer 188 400mg, sodium stearate 10mg, purified water 20ml in a 50mL beaker, stirred and dissolved in a 70°C water bath to form the water phase; the oil phase was injected into the water phase, and the temperature was 70°C and the stirring rate was 5000rpm shear Cut for 5 minutes, use an ultrasonic probe to sonicate for 5 minutes, and quickly place in an ice-water bath to cool to room temperature to obtain Ligustrazine Ferulic Acid Solid Lipid Nanoparticles (FATM-SLN).

Embodiment 2 The wavelength measurement of ligustrazine ferulic acid solid lipid nanoparticles of the present invention

Accurately weigh 0.0102g of FATM, place it in a 50mL volumetric flask, add methanol to dissolve it ultrasonically and make it to volume to obtain FATM stock solution A, precisely pipette 1mL of the stock solution into a 10mL volumetric flask, add methanol to dilute and make to volume, and then dilute with methanol It is blank control; Accurately draw FATM-SLN1mL prepared in Example 1, demulsify with methanol and settle to 25mL to obtain the test solution B; Accurately weigh all the auxiliary materials in the prescription, prepare SLN without FATM, as negative For sample C, accurately draw 1mL of the negative sample, break the emulsion with methanol and dilute to 25mL to obtain a blank solution; use a UV-visible spectrophotometer to scan the spectra of A, B, and C respectively at a wavelength of 200-500nm.

Accurately weigh 0.0142g of FATM and place it in a 50mL volumetric flask, dissolve it with methanol and constant volume, shake well to obtain a reference substance stock solution with a mass concentration of 0.284mg/mL, and accurately pipette the stock solution 0.1, 0.2, 0.4, 0.6, 0.8 , 1.0mL was placed in a 10mL volumetric flask, diluted to the mark with methanol, and shaken up to obtain a solution with a series of concentrations; the absorbance value (A) of the above solution was measured at a wavelength of 320nm, and the mass concentration (x) of the solution was taken as the abscissa, Take the absorbance value (y) as the ordinate, draw the standard curve and perform regression to get the standard line equation of FATM methanol solution: y=0.07501x-0.02971, r=0.9994, the linear range is 2.48～28.40μg/mL, the results are shown in the figure As shown in 1, it can be seen that the maximum absorption wavelength of FATM of the present invention is 320nm, and the auxiliary material has no absorption at the wavelength of 320nm, and has no interference to the determination of drugs.

The precision measurement of embodiment 3 ligustrazine ferulic acid solid lipid nanoparticles of the present invention

The methanol solution of ligustrazine ferulic acid solid lipid nanoparticles with 5.68, 11.36, 22.72 μg/mL low, medium and high concentrations of three concentrations was used to measure the precision, and the RSD was 0.24%, 0.94%, 0.55%, showing that the present invention The precision of ligustrazine ferulic acid solid lipid nanoparticles is good.

Embodiment 4 The mensuration of Ligustrazine Ferulic Acid Ligustrazine Solid Lipid Nanoparticle Recovery Rate of the present invention

Prepare 9 blank excipient samples according to the prescription quantity, add FATM at three levels of 80%, 100% and 120% of the prescription raw material respectively, mix well, configure 3 sample solutions for each content level, draw 1mL for each sample and fix with methanol Concentrate to 25mL, filter with a microporous membrane, measure its absorbance at a wavelength of 320nm to calculate the concentration, and the RSD is 0.72%, showing that the ligustrazine ferulic acid solid lipid nanoparticles recovery rate of the present invention is good.

Embodiment 5 Ligustrazine Ferulic Acid Ligustrazine Solid Lipid Nanoparticle Encapsulation Efficiency and Drug Loading Determination of the Present Invention

Fully swell the dextran particles with distilled water, pack the column, draw 1mL FATM-SLN onto the column, elute with distilled water, collect the eluate with opalescent part (FATM-SLN), and draw 0.5mL of this part to elute solution, add methanol to demulsify to 25mL, draw another 0.5mL FATM-SLN directly with methanol and demulsify to 25mL, measure the absorbance at 320nm according to the steps of Example 2, measure the content in the two samples respectively, according to the following The above formula was used to calculate the encapsulation efficiency and drug loading.

Encapsulation efficiency%=C _column /Ctotal×100%, wherein, C _column is _the amount of drug after FATM-SLN passes through the Sephadex gel, and Ctotal is the _total amount of FATM-SLN;

Drug loading %=C _package /C _plus × 100%, wherein, C _package is the amount of encapsulated drug, and C _plus is the total amount of drug added in the prescription; the results are shown in Table 1, it can be known that the solid fat of the present invention Plastid nanoparticles have higher encapsulation efficiency and smaller particle size, and the drug loading data show that the product has good stability in the room.

Table 1 Encapsulation efficiency and drug loading measurement results of different samples

Embodiment 6 Stability determination of ligustrazine ferulic acid solid lipid nanoparticles of the present invention

FATM-SLN was placed at 25°C and 4°C for 10 days, and the effect of temperature on the particle size of FATM-SLN was investigated; it was observed that white flocs appeared in different degrees in the three batches of samples placed at 25°C, RSD>3 %; The appearance of the sample placed at 4°C has no obvious change, RSD<2%.

Embodiment 7 Appearance, particle size distribution and Zeta potential measurement of ligustrazine ferulic acid solid lipid nanoparticles of the present invention

Use a transmission electron microscope to observe the surface morphology of FATM-SLN, dilute FATM-SLN with distilled water 10 times, take an appropriate amount dropwise on the copper grid covered with carbon film, stain with 1% phosphotungstic acid, leave it at room temperature until the film is formed, and use transmission electron microscopy Observed under a microscope, its shape is a spherical solid particle with a relatively round appearance and a particle size in the range of 100-200nm, as shown in Figure 2; absorb a certain amount of FATM-SLN, add ultra-pure water to dilute 10 times, and use a laser The particle size analyzer measures the particle size and potential. The average particle size is 105.3nm, the polydispersity index (PDI) is 0.254, and the Zeta potential is -34.8mV, as shown in Figure 3 and Figure 4.

Embodiment 8 Ligustrazine Ferulic Acid Solid Lipid Nanoparticles Drug Release Determination of the Present Invention

Precisely draw 1mL of FATM-SLN solution, put it into a dialysis bag, add 5mL of PBS (pH=7.4) into the dialysis bag, and place it in a 100mL jar containing 44mL of PBS. 1, 2, 4, 8, and 12 hours, take 3 mL of dialysis solution on time, filter and measure by UV method, and add 3 mL of fresh medium to the jar at the same time to calculate the cumulative release. The results are shown in Figure 5. You can see The release of FATM-SLN was the fastest at 0.51h, and the release amount reached 60.47% in 1h, and tended to be stable after 8h, and the drug was basically released completely.

Although the present invention has been described in detail with general descriptions and specific examples above, it is obvious to those skilled in the art that some modifications or improvements can be made on the basis of the present invention. Therefore, the modifications or improvements made on the basis of not departing from the spirit of the present invention all belong to the protection scope of the present invention.

Claims (7)

1. a kind of tetramethylpyrazine ferulate solid lipid nano granule, which is characterized in that by being formed comprising following parts by weight:

2. tetramethylpyrazine ferulate solid lipid nano granule according to claim 1, which is characterized in that by including following weight Part composition:

3. the preparation method of tetramethylpyrazine ferulate solid lipid nano granule according to claim 1 or claim 2, which is characterized in that packet Containing following steps:

Tetramethylpyrazine ferulate, egg yolk lecithin, butyl acetate, glycerin monostearate are weighed in 50mL's according to above-mentioned parts by weight In beaker, oily phase is dissolved as in 65-75 DEG C of water-bath；PLURONICS F87, the odium stearate, purified water of above-mentioned parts by weight are weighed again In the beaker of 50mL, water phase is dissolved as in 65-75 DEG C of stirring in water bath；The oil is mutually injected in the water phase, is in temperature 65-75 DEG C is that 4500-5500rpm shears 5min with stirring rate, is ultrasonically treated 5min using ultrasonic probe, is immediately placed in ice water Be cooled to room temperature in bath to get.

4. the preparation method of tetramethylpyrazine ferulate solid lipid nano granule according to claim 3, which is characterized in that described Water-bath solution temperature is 70 DEG C.

5. the preparation method of tetramethylpyrazine ferulate solid lipid nano granule according to claim 3, which is characterized in that described Stirring rate is 5000rpm.

6. the preparation method of tetramethylpyrazine ferulate solid lipid nano granule according to claim 3, which is characterized in that described Ultrasonic probe power accounting is 30%.

7. tetramethylpyrazine ferulate solid lipid nano granule as claimed in claim 1 or 2 is in preparation treatment ischemic cerebrovascular disease Application in drug.