CN103417479A - Ginsenoside Rg3 liposome and preparation method thereof - Google Patents

Abstract

The invention provides a ginsenoside Rg3 liposome and a preparation method thereof. The liposome and proliposome are used to encapsulate the drug, and the obtained liposome has a high encapsulation rate and stable properties, and can significantly increase the ginsenoside Rg3 liposome. The absorbability and bioavailability of Rg3, while enhancing its targeting to tumor tissue, improves drug efficacy. At the same time, it can be administered in various ways such as oral administration, injection, and pulmonary inhalation, so as to improve the scope of application of the drug and the compliance of patients.

Description

A kind of ginsenoside Rg3 liposome and preparation method thereof

technical field

The invention relates to the technical field of medicine, and in particular provides a ginsenoside Rg3 liposome, a preparation method thereof, and an application as a medicine and a health care product.

Background technique

Ginsenoside Rg3 is a tetracyclic triterpene saponin present in the natural medicine ginseng, with a molecular formula of C ₄₂ H ₇₂ O ₁₃ and a relative molecular weight of 784. Ginsenoside Rg3 has the effect of inhibiting tumor growth, mainly acts on the G2/M phase of the cell proliferation cycle, induces tumor cell apoptosis, selectively inhibits tumor cell adhesion and infiltration, resists tumor metastasis, and inhibits tumor angiogenesis. Regulate the body's immune function and so on.

6)ug/L。这可能是由于人参皂苷Rg3极难溶于水，从而导致口服吸收效率低，限制了其药效的发挥。 But ginsenoside Rg3 human body pharmacokinetic research finds, plasma concentration is very low after oral administration, and the maximum blood drug concentration value recorded after oral administration of 3.2mg/kg is only (16

6)ug/L. This may be due to the fact that ginsenoside Rg3 is extremely poorly soluble in water, resulting in low oral absorption efficiency, which limits its efficacy.

Liposomes (also known as lipid globules, liposomes) refer to microvesicles formed by encapsulating drugs in lipid bilayers. Liposome is a new type of passive targeting agent. After entering the body, it is easily taken up by the reticuloendothelial system (such as liver, spleen, and bone marrow) and quickly swallowed by macrophages as foreign bodies. Through endocytosis, liposomes can specifically concentrate drugs in the functional cell compartment, and also allow drugs that cannot pass through the serous membrane to reach lysosomes, and be digested and released drugs quickly after entering lysosomes, which can Maintain high drug concentrations in these target tissues. In addition, liposomes have little toxic and side effects on the body, and their lipid bilayer has greater similarity and tissue compatibility with biological membranes, and is easy to be absorbed by tissues. Liposome encapsulation of drugs is a physical process that does not change the molecular structure of the drug. Encapsulating the drug into liposomes can reduce renal excretion and metabolism and prolong the residence time of the drug in the blood, making the drug slowly released in the body, thereby prolonging the life of the drug. The action time of the drug can be shortened, the dosage can be reduced, and the side effects can be reduced.

At the same time, regardless of the size of the molecular weight of the drug, it can be encapsulated by the liposome. As a carrier, liposomes have advantages in delivering some anticancer drugs. Liposomes can not only improve the solubility of drugs by encapsulating poorly soluble drugs, but also change the pharmacological effects of drugs by targeted drug delivery. Because of the characteristics of the liposome composition, it can promote the clearance of liposomes by the reticuloendothelial system, which can target liposomes to a variety of cancer tissues. In order to facilitate the storage of liposomes before hydration, they can also be made into proliposomes, and proliposomes with good fluidity are also conducive to the preparation of solid preparations such as tablets and capsules.

The preparation process of the liposome is relatively simple and is suitable for industrial scale production. However, the encapsulation efficiency of the drug is significantly affected by the type of phospholipid used and the preparation method, and there is often a significant gap in stability. Therefore, comprehensive screening of phospholipids and preparation methods is required when encapsulating drugs. In terms of the encapsulation rate, stability and drug leakage rate of the preparation, the comprehensive optimization is achieved. The key to the preparation of liposomes lies in a suitable drug-to-lipid ratio and suitable adjuvant materials, so that the finally obtained liposomes can reach the standard of clinical application in terms of encapsulation efficiency, stability and drug loading.

At present, there is no related patent on making ginsenoside Rg3 into liposome or proliposome.

Contents of the invention

The invention provides a ginsenoside Rg3 liposome, which has high encapsulation efficiency and stable properties, and can significantly improve the absorbability and bioavailability of the ginsenoside Rg3.

The method also provides a ginsenoside Rg3 proliposome, which has high encapsulation efficiency and stable properties, and can significantly improve the absorbability and bioavailability of the ginsenoside Rg3.

The invention also discloses a preparation method of the above-mentioned ginsenoside Rg3 liposome and proliposome, which is suitable for industrial production.

The ginsenoside Rg3 liposome disclosed by the invention is characterized in that it is made by weight ratio of the following raw materials:

1 part of ginsenoside Rg3, 10-20 parts of phospholipids, 2-10 parts of cholesterol;

The phospholipid is selected from soybean lecithin or egg yolk lecithin.

The preparation method of described ginsenoside Rg3 liposome comprises the following steps:

1) Completely dissolve phospholipids, cholesterol, and ginsenoside Rg3 in n-butanol, use 50-100mL of n-butanol for each portion of ginsenoside, and spin-evaporate under reduced pressure at 30-50 degrees Celsius to form a film;

2) Vacuum drying for 24 hours will completely volatilize and remove n-butanol;

3) Add phosphate buffer solution with a pH of 6.0-7.5, add 20 mL of phosphate buffer solution to each portion of ginsenoside Rg3, and hydrate at 40 degrees Celsius for 30 minutes to obtain ginsenoside Rg3 liposome suspension;

4) The obtained ginsenoside Rg3 liposome suspension was subjected to high-pressure homogenization for 3 cycles, and then extruded twice through an extruder 100nm polycarbonate membrane to obtain Liposome suspension with an average particle size of 100nm.

The preparation method of described ginsenoside Rg3 liposome comprises the following steps:

1) Dissolve phospholipids, cholesterol, and ginsenoside Rg3 in absolute ethanol, and use 50-100 mL of ethanol for each portion of ginsenoside;

2) Inject the absolute ethanol solution into the constant temperature pH6.0-7.5 phosphate buffer solution at a constant speed of 30-50 degrees Celsius, and use 20-40mL phosphate buffer solution for each part of ginsenoside Rg3

3) Continue stirring for 2 hours to obtain a ginsenoside Rg3 liposome suspension;

4) The obtained ginsenoside Rg3 liposome suspension was subjected to high-pressure homogenization for 3 cycles, and then extruded twice through an extruder 100nm polycarbonate membrane to obtain Liposome suspension with an average particle size of 100nm.

5) Concentrating the obtained liposome suspension through an ultrafiltration membrane with a molecular weight cut-off of 5000 to remove ethanol.

The ginsenoside Rg3 proliposome disclosed by the invention is characterized in that it is made of the following raw materials by weight ratio:

1 part of ginsenoside Rg3, 10-20 parts of phospholipids, 2-10 parts of cholesterol, 4-8 parts of sorbitol;

Ginsenoside Rg3 proliposome mentioned in the present invention can adopt following method to prepare:

Spray drying: the ginsenoside Rg3 liposome suspension is mixed evenly with the water-soluble carrier solution in an appropriate proportion, and the ginsenoside Rg3 proliposome can be prepared by spray drying.

Spray drying conditions: inlet temperature 120 degrees Celsius, sample injection flow rate 10mL/min, atomization pressure 140kPa, wind speed 0.5-1m ³ /min.

Freeze-drying: mix the ginsenoside Rg3 liposome suspension and the sorbitol solution evenly according to the ratio, and freeze-dry to prepare the ginsenoside Rg3 proliposome.

The lipid and sorbitol were formulated into a solution according to the ratio, and were divided into 5ml vials, 2ml per bottle, pre-frozen at -80 degrees Celsius for 12 hours, and vacuum freeze-dried for 48 hours to obtain proliposomes.

The shape of the liposome prepared by the above ratio is uniform, the particle size distribution range is narrow, the average particle size distribution is 100～140nm, the encapsulation is more than 80%, the stability and the drug loading can reach the clinical application requirements; the preparation according to the above ratio After rehydration of the obtained proliposomes, the obtained liposomes have a uniform shape, a narrow particle size distribution range, an average particle size distribution of 100-150nm, an encapsulation of more than 80%, stability, and drug loading. Meet the requirements of clinical application.

The positive progress effect of the ginsenoside Rg3 liposome mentioned in the present invention is: for the insoluble drug Rg3 found suitable dosage form, and screened out the phospholipid adjuvant that is applicable to Rg3, simultaneously to prescription composition and preparation The process has been optimized, and the average particle size of the obtained liposomes is 100-140nm, which is a dual stable system of thermodynamics and kinetics. At the same time, the drug loading capacity is above 80%, which meets the requirements of the Pharmacopoeia. The liposome leakage rate is low, and the drug It is not easy to be lost, so as to ensure that the drug has a higher concentration and action time in the action area. Moreover, the liposome can be further developed into a proliposome, thereby expanding its route of administration, and is suitable for different drug users. At the same time, the preparation process is simple and easy, economical and reliable, and it is a practical new dosage form of Rg3.

The ginsenoside Rg3 liposome of the present invention can be administered in various ways and dosage forms, including intravenous injection, intraperitoneal injection, intramuscular injection, subcutaneous injection, intratumoral injection, oral administration, and pulmonary inhalation. For administration, specific dosage forms may include powder injection, liposome suspension for injection, injection, or infusion, oral suspension, soft capsule, aerosol and spray.

The ginsenoside Rg3 proliposome of the present invention can adopt multiple administration methods including oral administration, injection, pulmonary inhalation, and its dosage form can be tablet, enteric-coated tablet, hard capsule, soft capsule , Granules, Powder Inhalation, Powder Injection.

The encapsulation rate of the ginsenoside Rg3 proliposome prepared by the invention is greater than 80%, the property is stable, the preparation method is simple and convenient, and it is suitable for industrial scale-up production.

The positive effects of the present invention are: the present invention adopts liposomes and proliposomes to encapsulate medicines, and the obtained liposomes have high encapsulation efficiency and stable properties, and can significantly improve the absorbability and biological properties of ginsenoside Rg3. Utilization, and at the same time enhance its targeting to tumor tissue, improve drug efficacy. At the same time, it can be administered in various ways such as oral administration, injection, and pulmonary inhalation, so as to improve the scope of application of the drug and the compliance of patients.

Description of drawings

Figure 1. Curve of drug concentration in plasma versus time;

Figure 2 Optical microscope photo of ginsenoside Rg3 liposome (magnified 200 times);

Fig. 3 Dissolution curves of ginsenoside Rg3 proliposome tablet and ginsenoside Rg3 bulk drug.

Detailed ways

The present invention will be described in detail below using examples, but the present invention is not limited to the following examples.

Example 1

Weigh 0.5g ginsenoside Rg3, 7.5g egg yolk lecithin, and dissolve 1.25g cholesterol in 50ml n-butanol, place the solution in a pear-shaped bottle, evaporate under reduced pressure above the rotary evaporation to remove n-butanol, and make the phospholipids in the bottle A uniform film was formed on the wall, and the organic solvent was completely volatilized and removed by vacuum drying for 24 hours, and 10ml of 10mM pH6.5 phosphate buffer solution was added to fully hydrate at the phospholipid phase transition temperature to obtain a ginsenoside Rg3 liposome suspension. The obtained liposome suspension was homogenized for 3 cycles through a high-pressure homogenizer, and then granulated through a 100nm polycarbonate membrane under high pressure through an extruder, and the encapsulation efficiency of the finally gained liposomes was 87.3%, the average particle size is 131nm.

Example 2

Weigh 0.5g ginsenoside Rg3, 5g soybean lecithin, and dissolve 1.25g cholesterol in 50ml ethanol, slowly inject the phospholipid ethanol solution into 20mL of 20mM pH6.5 phosphate buffer solution shaken in a constant temperature water bath at 50 degrees Celsius with a micro-syringe, to obtain Ginsenoside Rg3 liposome suspension, the obtained liposome suspension is homogenized through a high-pressure homogenizer for 3 cycles, and then granulated through a 100nm polycarbonate membrane under high pressure by an extruder, An ultrafiltration membrane with a molecular weight cut-off of 5000 was selected, and ethanol was removed by ultrafiltration and concentration. The encapsulation efficiency of the finally obtained liposome was 81.2%, and the average particle size was 136nm.

Example 3

Weigh 0.5g ginsenoside Rg3, 7.5g egg yolk lecithin, and dissolve 1.25g cholesterol in 50ml ethanol, slowly inject the phospholipid ethanol solution into 20mL of 10mM pH6.5 phosphate buffer solution shaken in a constant temperature water bath at 50 degrees Celsius with a micro syringe, To obtain the ginsenoside Rg3 liposome suspension, the obtained liposome suspension was homogenized by a high-pressure homogenizer for 3 cycles, and then passed through a 100nm polycarbonate membrane for 2 times under high pressure by an extruder , select the ultrafiltration membrane with molecular weight cut off 5000, remove ethanol with the mode of ultrafiltration concentration, finally gained liposome encapsulation rate is 81.2%, and average particle diameter is 105nm.

Example 4

Weigh 0.5g ginsenoside Rg3, 10g soybean lecithin, and dissolve 1.25g cholesterol in 50ml ethanol, and slowly inject the phospholipid ethanol solution into 20mL of 10mM pH6.5 phosphate buffer solution shaken in a constant temperature water bath at 50 degrees Celsius with a micro-syringe to obtain Ginsenoside Rg3 liposome suspension, the obtained liposome suspension is homogenized through a high-pressure homogenizer for 3 cycles, and then granulated through a 100nm polycarbonate membrane under high pressure by an extruder, An ultrafiltration membrane with a molecular weight cut-off of 5000 was selected, and ethanol was removed by ultrafiltration and concentration. The encapsulation efficiency of the finally obtained liposome was 81.2%, and the average particle size was 137nm.

Example 5

Weigh 0.5g ginsenoside Rg3, 5g egg yolk lecithin, and dissolve 1.25g cholesterol in 50ml n-butanol, place the solution in a pear-shaped bottle, evaporate under reduced pressure above the rotary evaporation to remove n-butanol, and make the phospholipids on the bottle wall Form a uniform film on the surface, dry in vacuum for 24h, remove the organic solvent completely, add 15ml 10mM pH6.5 phosphate buffer solution to fully hydrate on the phospholipid phase transition temperature, obtain ginsenoside Rg3 liposome suspension, and the obtained lipid The body suspension was homogenized by a high-pressure homogenizer for 3 cycles, and then granulated by a 100nm polycarbonate film under high pressure through an extruder for 2 times, and then an aqueous solution with 2g of sorbitol was added, mixed evenly, and sprayed. Dry to obtain powdered ginsenoside Rg3 proliposome. The encapsulation efficiency is 86.4%, and the average particle size is 101nm. The proliposome powder is filled into a hard capsule to obtain a hard capsule.

Example 6

Weigh 0.5g ginsenoside Rg3, 7.5g egg yolk lecithin, and dissolve 1.25g cholesterol in 50ml n-butanol, place the solution in a pear-shaped bottle, evaporate under reduced pressure above the rotary evaporation to remove n-butanol, and make the phospholipids in the bottle A uniform film is formed on the wall, and the organic solvent is completely volatilized and removed by vacuum drying for 24 hours. Add 15ml of 10mM pH6.5 phosphate buffer solution to fully hydrate on the phospholipid phase transition temperature to obtain a ginsenoside Rg3 liposome suspension. The plastid suspension was homogenized by a high-pressure homogenizer for 3 cycles, and then passed through a 100nm polycarbonate membrane under high pressure by an extruder for 2 times of sizing, adding an aqueous solution dissolved in 4g of sorbitol, and passing through a 0.22μm filter membrane Sterilize by filtration, divide into vials, and freeze-dry to obtain ginsenoside Rg3 proliposome powder injection. The encapsulation efficiency is 83.7%, and the average particle size is 147nm.

Experimental example 1

Weigh 0.5g ginsenoside Rg3, 6g soybean lecithin, and dissolve 1.5g cholesterol in 50ml n-butanol, place the solution in a pear-shaped bottle, evaporate under reduced pressure above the rotary evaporation to remove n-butanol, and make the phospholipids on the bottle wall Form a uniform film on the surface, dry in vacuum for 24 hours, and remove the organic solvent completely. Add 15ml of 10mM pH6.5 phosphate buffer solution to fully hydrate at the phase transition temperature of phospholipids to obtain ginsenoside Rg liposome suspension. Alcohol aqueous solution, mixed evenly, and spray-dried to obtain powdered ginsenoside Rg3 proliposome.

The proliposome powder is mixed with micropowder silica gel, cross-linked carboxymethyl cellulose, and cross-linked starch, and compressed into tablets to prepare oral tablets. According to the Pharmacopoeia, the dissolution rate is compared with that of the ginsenoside Rg3 raw material drug, and the results are shown in Figure 2. Data shows that after stripping 4h, the drug dissolution rate of proliposome reaches more than 80%, and the dissolution rate of ginsenoside Rg3 crude drug is almost none. It can be confirmed that the dissolution rate of the drug can be significantly improved after the ginsenoside Rg3 is prepared as a proliposome.

The oral tablet can be coated with an enteric coating to obtain an enteric tablet.

Example 7

Put the ginsenoside Rg3 proliposome prepared in Example 4 into a quantitative powder inhalation device to obtain a powder inhaler. The average aerodynamic particle size of the powder is 5 μm, which is suitable for pulmonary administration.

Example 8

Put the ginsenoside Rg3 liposome prepared in Example 1 into the aerosol device together with the propellant to obtain the ginsenoside Rg3 liposome aerosol.

Example 9

After the ginsenoside Rg3 liposomes prepared in Examples 1 and 2 were sterilized by filtration through a 0.22 μm filter membrane, they were dispensed into vials under aseptic conditions to obtain ginsenoside Rg3 liposome injections. The injection is suitable for intramuscular injection, subcutaneous injection and intratumoral injection.

Show advantage of the present invention by following experiment:

Test example 1

Effect of different phospholipid materials on liposome encapsulation efficiency

experiment material

Soy lecithin, egg yolk lecithin, dipalmitoylphosphatidylcholine, distearoylphosphatidylcholine, dipalmitoylphosphatidylethanolamine, dipalmitoylphosphatidic acid, ginsenoside Rg3, cholesterol;

The liposome prepared by embodiment 1, embodiment 2;

Control group 1: ginsenoside Rg3: dipalmitoylphosphatidylcholine: cholesterol;

Control group 2: ginsenoside Rg3: distearoylphosphatidylcholine: cholesterol;

Control group 3: ginsenoside Rg3: dipalmitoylphosphatidylethanolamine: cholesterol;

Control group 4: Ginsenoside Rg3: dipalmitoylphosphatidic acid: cholesterol;

The amount of raw materials added in each of the above groups is the mass ratio, the ratio is ginsenoside Rg3:phospholipid:cholesterol=1:15:2.5;

Encapsulation efficiency is an important index to investigate the properties of liposomes, and the low encapsulation efficiency of liposomes to drugs is a major problem that liposomes need to overcome. In the experiment, it was found that the type of phospholipid had a significant impact on the encapsulation efficiency of liposomes. The liposome suspension was prepared by thin film dispersion, and the effect of phospholipid quality on the encapsulation efficiency of liposomes was investigated by single factor experiments. The results are shown in Table 1:

The influence of table 1 different phospholipids on liposome encapsulation efficiency and particle size

Ginsenoside Rg3: Phospholipid: Cholesterol (w/w) Particle size (nm) Encapsulation rate (%) Example 1 131 87.2 Example 2 136 88.7 Control group 1 125 52.1 Control group 2 105 39.5 Control group 3 111 65.3 Control group 4 135 62.8

Conclusion: It can be seen from the table that soybean lecithin and egg yolk lecithin have a better encapsulation effect on ginsenoside Rg3, which can reach the encapsulation rate of more than 80% required by the Pharmacopoeia, but dipalmitoylphosphatidylcholine, di Stearoylphosphatidylcholine, dipalmitoylphosphatidylethanolamine, and dipalmitoylphosphatidic acid have relatively weak encapsulation ability to ginsenoside Rg3, which is due to the phospholipid molecular structure of dipalmitoyls and distearoyls. The higher saturation of Rg3 is not conducive to the insertion of Rg3, thus reducing the encapsulation efficiency.

Test example 2

Effect of Phospholipid Amount on Encapsulation Efficiency

In the experiment, it was found that the amount of phospholipids is a key factor affecting the quality of liposomes. The liposome suspension was prepared by film dispersion, and the effect of the amount of phospholipids on liposome particle size and encapsulation efficiency was investigated through single factor experiments.

The liposome prepared by embodiment 2, embodiment 3, embodiment 4;

Control group 1: ginsenoside Rg3: soybean lecithin: cholesterol 1: 0.5: 2.5;

Control group 2: ginsenoside Rg3: egg yolk lecithin: cholesterol 1:5:2.5;

Control group 3: ginsenoside Rg3: egg yolk lecithin: cholesterol 1:25:2.5;

Control group 4: ginsenoside Rg3: egg yolk lecithin: cholesterol 1:30:2.5;

The results are shown in Table 2:

The influence of table 2 phospholipid dosage on liposome particle size and encapsulation efficiency

Ginsenoside Rg3: Phospholipid: Cholesterol (w/w) Particle size (nm) Encapsulation rate (%) Control group 1 174 20.1 Control group 2 125 45.8 Example 2 136 82.9 Example 3 105 87.3 Control group 4 111 90.4 Control group 3 135 88.6 Control group 4 124 87.2

Summary: It can be seen from the table that the encapsulation efficiency of liposomes is significantly affected by the amount of phospholipids. When the drug-to-lipid ratio is greater than 1:10, the encapsulation efficiency is lower than 80%, which does not meet the requirements of the Pharmacopoeia for liposome drugs 80% % or more, with the increase of phospholipid dosage, the encapsulation efficiency will increase. When the drug-lipid ratio is less than 1:20, although the encapsulation efficiency can reach more than 80%, the drug loading is too low to meet the clinical requirements. Require. At the same time, it is found that when the amount of phospholipids is more than 20 parts, the viscosity of the system increases, resulting in a longer hydration time. The original 30min hydration is extended to more than 1h. The prolongation of the hydration time will inevitably lead to an increase in the amount of phospholipids being oxidized. It is toxic, which is detrimental to the safety of liposomes. At the same time, too high concentration of phospholipids will make it difficult for the suspension to be extruded by the extruder, and the drug-loaded phospholipids cannot pass through the polycarbonate membrane. Thereby, the recovery rate of the drug is decreased, which will cause the recovery rate of the drug to decrease by at least 20% after calculation, and it is advisable to use 10-20 parts of phospholipids in comprehensive consideration.

Test 3

Investigation of Drug Leakage Rate

An important indicator of liposome stability is the leakage rate of the drug. Appropriate amount of liposome obtained after hydration and reconstitution of ginsenoside Rg3 liposome and Rg3 proliposome was placed in a dialysis bag, and phosphate buffer was used as a release medium. Encapsulation rate, leakage rate calculation formula is as follows:

Q _drain = (EF ₁ -EF ₂ )/EF ₁ × 100%

EF ₁ is the initial encapsulation efficiency, and EF ₂ is the encapsulation efficiency after storage for 3 months.

Table 3 Ginsenoside Rg3 liposome leakage rate

Ginsenoside Rg3: Phospholipid: Cholesterol (w/w) Leakage rate (%) Example 1 5.3 Example 2 5.9 Example 3 6.8 Example 4 5.7

The leakage rate of the drug was 5.3-6.8% after 3 months of storage, and the leakage of the drug was very slow. The constructed liposome had a strong ability to encapsulate the drug to Rg3, and the stability could meet the clinical requirements.

Example 4

whole liposome

Usually the obtained liposome suspension is granulated by high-pressure homogenization or extrusion, but it is found in experiments that these two methods are used alone to carry out granulation effect to ginsenoside Rg3 liposomes, if Only applicable to the high-pressure homogenization method, the obtained particle size distribution is not uniform, the liposome membrane-passing ability is poor when only the extrusion method is used, and the recovery rate of the drug is low, below 40%, the obtained liposomes are suspended The liquid was homogenized by a high-pressure homogenizer for 3 cycles, and then granulated by a 100nm polycarbonate membrane under high pressure through an extruder. It was found that the obtained liposome particle size distribution was uniform and the distribution range was narrow. The recovery rate is improved and can reach more than 75%.

Select Examples 1 to 4 to investigate the influence of different granulation methods on the properties of liposomes, wherein A represents only high-pressure homogeneous sizing, B represents only extrusion sizing, and C represents high-pressure homogenization before passing Extrusion granulation.

the

Table 4 Effects of different granulation methods on liposome properties

Whole grain method Drug recovery rate (%) Particle size (nm) Example 1A 62 145～240 Example 2A 75 157～324 Example 3A 81 133～232 Example 4A 77 109～318 Example 1B 37 124 Example 2B 35 117 Example 3B 29 127 Example 4B 33 120 Example 1C 82 131 Example 2C 87 136 Example 3C 76 105 Example 4C 79 111

In the process of preparing proliposomes, it was found that the choice and amount of water-soluble carrier had a crucial influence on the construction of proliposomes and the properties of liposomes after reconstitution.

Carriers commonly used to prepare proliposomes include sorbitol, mannitol, lactose, sucrose, etc., among which sorbitol has a better effect. This is mainly because sorbitol has a porous structure, contains a large amount of phospholipids, and has better dispersion when hydrated. The water solubility of lactose is not as good as that of sorbitol, and the dispersibility is not good when it is hydrated, and there is a layering phenomenon after being placed. Mannitol is prone to wet condensation during the preparation process, so it is not suitable for the preparation of proliposomes by this method. Therefore, sorbitol was finally selected as the carrier material.

Test example 5

The amount of fixed ginsenoside Rg3, phospholipid, and cholesterol was 1:15:2.5. Taking sorbitol as an example, proliposomes were prepared, and the appearance and redispersibility of the proliposomes were investigated. After reconstruction, the liposomes were obtained Particle size and encapsulation efficiency, grouped as follows:

Example 5: Ginsenoside Rg3: soybean lecithin: cholesterol: sorbitol 1:15:2.5:4;

Example 6: Ginsenoside Rg3: egg yolk lecithin: cholesterol: sorbitol 1:15:2.5:8;

Control group 1: ginsenoside Rg3: soybean lecithin: cholesterol: sorbitol 1:15:2.5:2;

Control group 2: ginsenoside Rg3: egg yolk lecithin: cholesterol: sorbitol 1:15:2.5:4;

Control group 3: ginsenoside Rg3: soybean lecithin: cholesterol: sorbitol 1:15:2.5:24;

The results are shown in Table 3:

The consumption of table 5 sorbitol influences on the proliposome property

Ginsenoside Rg3: Phospholipids: Cholesterol: Sorbitol (w/w) Proliposome Appearance redispersibility Particle size (nm) Encapsulation rate (%) Control group 1 softening, collapse Difference 143 86.2 Control group 2 slightly softened, collapsed generally 114 87.5 Example 5 fluffy and fluid good 101 90.2 Example 6 fluffy and fluid good 147 88.4 Control group 3 fluffy and fluid good 192 87.7

It can be seen from Table 5 that the addition of the water-soluble carrier has a certain effect on the particle size and encapsulation efficiency of the proliposome hydration reconstructed liposome, but the effect is not significant. However, there was a significant impact on the redispersibility of the proliposomes during hydration reconstitution and the appearance of the proliposomes. When the amount of sorbitol is less than 4 parts by weight, the appearance of the proliposome is poor, softening and collapse occurs, aggregation occurs during the hydration reconstruction process, and redispersion is difficult. In order to increase the drug loading capacity of the system as much as possible, the dosage of the water-soluble carrier is selected to be 4-8 parts.

Test example 6

Liposomes improve drug efficacy

Pharmacokinetic study:

Self-control method was adopted, and 5 Wistar rats that had not been tested were used, and each rat was given a single dose of ginsenoside Rg3 liposome (dose 5 mg/kg). Fasting 12h before administration. Free eating and drinking were allowed 4h after administration. Draw blank blood before administration, and take 0.5 mL of blood into heparinized centrifuge tubes at 0.25, 0.5, 0.75, 1, 1.5, 2, 2.5, 3, 3.5, 4, 5, 6, 8, 10, and 12 hours after administration In the process, the plasma was separated by centrifugation at 4000rpm for 10min. The resulting plasma was stored at -20°C pending analysis. In the second week, each Wistar rat was given a single dose of ginsenoside suspension (dose 5mg/kg). The rest of the operations were the same as the experimental group.

See Tables 4 and 5 for the blood drug concentration-time curves and time point blood drug concentration measurement results for experimental animals after administration. The time-varying curve of drug concentration in plasma is shown in Figure 1 (-■-dissolution curve of ginsenoside Rg3 proliposomal tablet; -◆-dissolution curve of ginsenoside Rg3 raw drug). the

Table 6 Plasma Drug Concentration of Ginsenoside Suspension

time (h) 1 2 3 4 5 average value SD 0.25 2.61 12.31 10.57 8.45 52.69 17.326 20.10448 0.5 9.56 27.92 19.57 55.73 128.67 48.29 48.10428 0.75 138.62 84.08 44.87 60.15 21.41 69.826 44.71109 1 64.19 124.45 282.71 77.36 34.13 116.568 98.41498 1.5 73.51 207.67 404.24 185.91 116.69 197.604 127.3606 2 371.81 167.59 259.34 347.69 478.73 325.032 117.7158 2.5 54.72 141.99 78.95 216.42 276.73 153.762 92.99447 3 126.35 25.53 99.13 109.37 220.16 116.148 69.76224 3.5 102.19 43.72 56.18 65.01 15.16 56.452 31.75063 4 65.35 10.22 60.42 24.35 102.21 52.51 36.3282 5 77.57 53.56 48.21 34.14 8.72 44.44 25.38204 6 50.02 45.67 24.72 5.47 40.43 33.262 18.24507 8 22.03 16.8 14.16 37.44 40.22 26.13 11.97481 10 11.82 18.02 22.75 5 7.43 13.004 7.36003 12 1.14 7.78 10.22 2.32 18.12 7.916 6.83151

Table 7 Ginsenoside Rg3 liposome group

time (h) 1 2 3 4 5 average value SD 0.25 103.29 69.64 45.93 71.48 14.78 61.024 32.92535 0.5 261.48 121.67 101.77 213.43 75.32 154.734 79.12794 0.75 221.71 141.77 210.42 305.36 277.37 231.326 63.54307 1 218.73 367.44 347.41 381.77 492.72 361.614 97.78366 1.5 502.61 457.82 342.59 387.11 421.51 422.328 61.85468 2 318.52 441.55 406.74 327.53 349.69 368.806 53.21769 2.5 442.86 298.11 252.35 331.45 388.61 342.676 74.86271 3 196.19 309.67 335.77 275.34 372.48 297.89 67.06102 3.5 286.82 305.31 166.22 243.67 331.98 266.8 64.77574 4 341.21 97.49 127.45 225.61 282.34 214.82 102.5301 5 120.76 253.57 97.62 164.37 55.49 138.362 75.4765 6 120.79 151.11 24.62 64.43 50.39 82.268 52.15568 8 11.44 37.39 20.78 45.51 61.82 35.388 19.95002 10 13.14 21.32 9.42 31.37 5.57 16.164 10.30371 12 3.45 15.27 4.77 24.52 1.36 9.874 9.79067

From Table 4 and Table 5 and Figure 1 (--dissolution curve of ginsenoside Rg3 proliposome tablet; -◆-dissolution curve of ginsenoside Rg3 raw material drug), it can be seen that the blood drug concentration of ginsenoside Rg3 liposome reaches the peak The time T _max moved forward, and the maximum plasma concentration C _max increased significantly.

Compartment model pharmacokinetic parameters

The average plasma concentration data of ginsenoside Rg3 liposomes and commercially available ginsenoside Rg3 raw materials were analyzed with the 3P97 pharmacokinetic program, and then the average blood drug concentrations of liposomes and commercially available raw materials were analyzed using a compartment model. Fitting, AIC (Akaike's information criterion) value and R (correlation coefficient between fitting value and measured value) are used as indicators for comprehensive comparison. The smaller the AIC value and the larger the R value, the higher the fitting degree. After calculation, the pharmacokinetic parameters of liposomes and commercially available raw materials are shown in Table 6 below.

Table 8 Pharmacokinetic parameters of Rg3 liposomes and commercially available raw materials

parameter Rg3 liposome Commercial API A (μg/mL) 1171.256840 182.930450 Ke(1/h) 0.426852 0.270996 Ka(1/h) 1.004604 1.526343 Lag Time(h) 0.158983 0.172734 T1/2(ka)(h) 0.689970 0.454123 T1/2(ke)(h) 1.623859 2.557775 Tmax(h) 1.481451 2.176930 Cmax(μg/mL) 357.905460 103.596184 AUC(μg·h/mL) 1578.053710 555.180910 CL/F(s)(mg mL/(h μg)) 0.003168 0.009006 V/F(c)(mg·μg/mL) 0.007423 0.033233

It can be seen from Table 8 that the Tmax of Rg3 liposome is about 1.48h, the Tmax of the commercially available ginsenoside Rg3 bulk drug is about 2.18h, the Cmax of Rg3 liposome is about 357.90 μg/mL, and the Cmax of the bulk drug is about 103.60 μg/mL, the Tmax of Rg3 liposomes moved forward, and the Cmax increased significantly. The results showed that the blood drug concentration data of Rg3 liposome preparation and the commercially available drug substance all had the highest fitting degree with the seventh model (single-compartment first-order absorption, weighted as /lC ² ) in the 3P97 program.

relative bioavailability

Result With the average blood drug concentration-time area under the curve (AUC) data of above-mentioned measuring, calculate the relative bioavailability Fr of self-emulsifying dosage form to the commercially available sheet of same dose, calculation formula is:

Calculated by the area under the drug-time curve AUC, from the formula of relative bioavailability Fr(SEDDS)= 1578.053710/555.180910×100%=284.24%. Compared with the commercially available raw materials, the bioavailability of the Rg3 liposome preparation has been greatly improved.

The removal of the organic solvent does not use the conventional method of removing the organic solvent by heating and stirring, because this method takes a long time and increases the possibility of phospholipid oxidation. At the same time, the removal of the organic solvent is not complete, and it is easy to cause residue, which affects the quality of the product. safety. Therefore, in the present invention, an ultrafiltration membrane with a molecular weight cut-off of 5000 is selected to remove ethanol by means of ultrafiltration and concentration, which can easily make the ethanol residue below 400ppm to ensure the safety of the product.

Claims (5)

1. ginsenoside Rg3's liposome it is characterized in that being made by weight by following raw material:

1 part of ginsenoside Rg3,0～20 part of phosphatidase 11,2～10 parts, cholesterol

Described phospholipid is selected from soybean lecithin or Ovum Gallus domesticus Flavus lecithin.

2. the preparation method of ginsenoside Rg3's liposome as claimed in claim 1 comprises the following steps:

1) by phospholipid, cholesterol, the ginsenoside Rg3 is dissolved in n-butyl alcohol fully, and every part of ginsenoside uses 50～100mL n-butyl alcohol, decompression rotary evaporation film forming under 30～50 degrees centigrade;

2) vacuum drying 24h will have the n-butyl alcohol removal of volatilizing fully;

3) add the phosphate buffered solution of pH6.0-7.5, every part of ginsenoside Rg3 adds the 20mL phosphate buffered solution, and 40 degrees centigrade of aquation 30min obtain ginsenoside Rg3's liposome turbid liquor;

4) by the ginsenoside Rg3's liposome turbid liquor obtained, the liposome turbid liquor that obtains, through 3 circulations of high pressure homogenization, then is extruded to granulate 2 times through extruding instrument 100nm polycarbonate membrane, obtain the liposome turbid liquor of mean diameter at 100nm.

3. the preparation method of ginsenoside Rg3's liposome as claimed in claim 1 comprises the following steps:

1) by phospholipid, cholesterol, the ginsenoside Rg3 is dissolved in dehydrated alcohol, and every part of ginsenoside uses 50～100mL ethanol;

2) ethanol solution at the uniform velocity is injected under 30～50 degrees centigrade in constant temperature pH6.0-7.5 phosphate buffered solution, every part of ginsenoside Rg3 uses 20mL～40mL phosphate buffered solution;

3) continue to stir 2h and obtain ginsenoside Rg3's liposome turbid liquor;

4) by the ginsenoside Rg3's liposome turbid liquor obtained, the liposome turbid liquor that obtains, through 3 circulations of high pressure homogenization, then is extruded to granulate 2 times through extruding instrument 100nm polycarbonate membrane, obtain the liposome turbid liquor of mean diameter at 100nm;

5) liposome turbid liquor obtained is removed to ethanol through the ultrafilter membrane ultrafiltration and concentration of molecular cut off 5000, obtain.

4. ginsenoside Rg3's pro-liposome it is characterized in that being made by weight by following raw material:

1 part of ginsenoside Rg3,0～20 part of phosphatidase 11,2～10 parts, cholesterol, 4～8 parts of sorbitol.

5. the preparation method of ginsenoside Rg3's pro-liposome as claimed in claim 4 comprises the following steps:

Ginsenoside Rg3's liposome turbid liquor claimed in claim 2 is mixed with sorbitol, and spray-dried, lyophilization can make ginsenoside Rg3's pro-liposome;

1) spray drying condition: 120 degrees centigrade of inlet temperatures, sample introduction flow velocity 10mL/min, atomizing pressure 140kPa, wind speed 0.5-1m3/min;

2) lyophilization condition: ginsenoside Rg3's liposome turbid liquor and sorbitol are mixed and made into to solution, are sub-packed in the 5ml cillin bottle, every bottle of 2ml, in-80 degrees centigrade of pre-freeze 12h, vacuum lyophilization 48h, obtain pro-liposome.

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