WO2018133108A1

WO2018133108A1 - Ziyuglycoside ii emulsion and preparation method therefor

Info

Publication number: WO2018133108A1
Application number: PCT/CN2017/072228
Authority: WO
Inventors: 杨世林
Original assignee: 四川英路维特医药科技有限公司
Priority date: 2017-01-23
Filing date: 2017-01-23
Publication date: 2018-07-26

Abstract

A ziyuglycoside II emulsion, a preparation method therefor and a use thereof. Raw and auxiliary materials of the emulsion comprises: 1 part of ziyuglycoside II, 1-350 parts of an oil phase, and 0.5-40 parts of an emulsifier. Also provided are the usages of the emulsion in preparing medicines for treating and/or preventing myelosuppression, and in preparing medicines for increasing the count of blood cells and hemoglobin.

Description

Earthworm saponin II emulsion and preparation method thereof

Technical field

The invention relates to a saponin II emulsion and a preparation method thereof, and belongs to the field of medicine.

Background technique

Myelosuppression is a clinically common hematopoietic disease that can occur in radiation therapy and/or chemotherapy of various systemic neoplastic diseases, radiation damage caused by ionizing radiation, viral hepatitis, parvovirus infection or drugs (chloramphenicol). , benzene, sulfonamides, anti-epileptic drugs, sedatives, anti-thyroid drugs, anti-diabetes drugs, anti-malaria, sleeping pills and other factors. Myelosuppression can cause damage to the bone marrow microenvironment, hematopoietic stem cells, hematopoietic growth factors, etc., and the granulosa, red, and megakaryocyte systems are inhibited. Lack of granulocytes can cause serious infections; a marked reduction in red blood cells can cause severe anemia; a marked drop in platelets causes severe bleeding and even death. At present, there is still no effective treatment for bone marrow suppression in the clinic, and it is urgent to develop a therapeutic drug with better efficacy.

Diltiazem II, chemical name: 3-O-α-L-arabino-19-carboxylic acid (ziyu-glycoside II), is a mantle from the genus Rosaceae or A pharmacologically active compound is extracted from the roots of the longleaf mantle. CN101119740A discloses the use of saponin II in the preparation of a medicament for increasing red blood cells and hemoglobin. However, in actual use, it has been found that saponin II is not effective, and it is often difficult to obtain a better blood cell level and a bone marrow suppression treatment when used alone. Therefore, there is an urgent need to improve the efficacy of the saponin II and promote the clinical application of the drug.

Summary of the invention

It is an object of the present invention to provide a saponin II emulsion and a process for the preparation thereof.

The present invention provides a saponin II emulsion prepared by adding a raw material of the following weight ratio: 1 part of saponin II, 1-350 parts of oil phase, 0.5-40 parts of emulsifier.

Further, it is prepared by including a raw material of the following weight ratio: 1 part of saponin II, 300 parts of oil phase, and 20 parts of emulsifier.

Further, the oil phase is selected from the group consisting of soybean oil, medium chain triglyceride, fish oil, olive oil, ethyl oleate, castor oil, or a mixture of two or more; the emulsifier is selected from the group consisting of egg yolk eggs. One or two of phospholipids and soybean phospholipids.

Further, it further comprises 0.5 to 5 parts of a stabilizer and 1 to 10 parts of an isotonicity adjusting agent.

Further, the stabilizer is oleic acid; and the isotonicity adjusting agent is glycerin.

Further, it is prepared from the following raw materials by weight ratio: 1 part of saponin II, 300 parts of soybean oil, 20 parts of soybean phospholipid, 0.5 part of oleic acid, 1 part of glycerin, and water is added to the saponin II. The concentration was 0.3 mg/mL.

The invention provides a preparation method of the emulsion, comprising the following steps:

a, weigh each weight ratio of saponin II, oil phase and emulsifier, dissolved in organic solvent, mixed solution for use;

b, removing the organic solvent in the mixed solution of step a, and then adding a stabilizer, glycerin and water;

c, shear, homogenize, sterilize, that is.

Further, the organic solvent is ethanol; the shearing condition is: shearing at 10,000 rpm for 5 min; and the homogenizing condition is: homogenizing 4 times under a pressure of 800 bar.

The invention provides the use of the emulsion in the manufacture of a medicament for the treatment and/or prevention of myelosuppression.

The present invention provides the use of the emulsion in the manufacture of a medicament for increasing the number of blood cells and hemoglobin.

The present invention provides a method of treating and/or preventing myelosuppression using the emulsion.

The present invention provides a method of raising the number of blood cells and hemoglobin using the emulsion.

During the research, the inventors found that the reason that the effect of the saponin II on raising the blood cell level is poor is that the solubility is low and the gastrointestinal absorption rate is small, resulting in low bioavailability of the drug and limiting the exertion of its efficacy. The invention adopts a specific kind and proportion of auxiliary materials, and can prepare the saponin II as a better quality emulsion. In the efficacy experiment, compared with the model group, the saponin II emulsion of the present invention can significantly increase the number of peripheral blood WBC, RBC, PLT, NEUT and HGB, and the drug effect is obviously superior to that of the saponin II drug, indicating the invention The preparation of the saponin II as an emulsion can improve the bioavailability of the main drug, enhance its blood cell number, and prevent bone marrow suppression.

It is apparent that various other modifications, substitutions and changes can be made in the form of the above-described embodiments of the present invention.

The above content of the present invention will be further described in detail below by way of specific embodiments in the form of embodiments. However, the scope of the above-mentioned subject matter of the present invention should not be construed as being limited to the following examples. Any technique implemented based on the above description of the present invention is within the scope of the present invention.

DRAWINGS

Figure 1 is a graph comparing the counts of bone marrow hematopoietic stem cells in mice of each experimental group.

detailed description

The raw materials and equipment used in the specific embodiments of the present invention are known products and are commercially available through purchase. Product acquisition.

Example 1 Preparation of the saponin II emulsion of the present invention

Prescription one (A): saponin II 0.6g, soybean oil 180g, egg yolk lecithin 12g, glycerin 0.6g, water added to 2000ml

Prescription 2 (B): Diosgenin II 0.6g, medium chain triglyceride 120g, egg yolk lecithin 8g, glycerol 0.6g, water added to 2000ml

Prescription III (C): saponin II 0.6g, fish oil 60g, egg yolk lecithin 4g, glycerol 0.6g, water added to 2000ml

Prescription IV (D): saponin II 0.6g, olive oil 6g, egg yolk lecithin 1g, glycerol 0.6g, water added to 2000ml

Prescription 5 (E): Diosgenin II 6g, ethyl oleate 300g, egg yolk lecithin 30g, glycerol 5g, water added to 20000ml

Prescription six (F): saponin II 9g, castor oil 300g, egg yolk lecithin 30g, glycerin 9g, water added to 30,000ml

Prescription seven (G): saponin II 9g, soybean oil 300g, soybean phospholipid 30g, glycerol 10g, water added to 30,000ml

The preparation method comprises the following steps: mixing the saponin II, the oil and the phospholipid, dissolving in ethanol, and removing the ethanol by rotary evaporation under reduced pressure, and adding the oil according to the weight ratio of the saponin II: oleic acid: glycerin 1:0.5:1. Acid, glycerin, adding water to the emulsion, the concentration of saponin II is 0.3mg/mL, high-speed shearing at 10000rpm for 5min, high pressure homogenization at 800bar pressure for 4 times, autoclaving, that is.

The beneficial effects of the present invention are exemplified below by experiments.

Experimental Example 1 Quality Evaluation of Preparation of Diosgenin II Emulsion Using Different Excipients

In this experiment, 9 experimental groups were set up. Take the saponin II 0.6g, the oil phase, emulsifier, stabilizer and isotonicity adjuster as shown in Table 1, and then the ratio of the saponin II, oil phase and emulsifier to 1:300 by weight: 20 mixed, dissolved in ethanol, rotary evaporation under reduced pressure of ethanol, according to the weight ratio of saponin II.. Stabilizer: isotonicity regulator 1:0.5:1 high-speed stirring to add stabilizer, isotonic regulator, add water to The concentration of saponin II in the emulsion was 0.3 mg/mL, high-speed shearing at 10,000 rpm for 5 min, high pressure homogenization at 800 bar pressure for 4 times, and autoclaving. Observe the appearance of delamination, flocculation, sticking to the wall, determine the particle size, determine Ke (determine the absorbance A _o and A before and after centrifugation of the emulsion by spectrophotometry to calculate the stability constant Ke = (A _o -A) / A _o ×100%. The smaller the Ke value, the less the oil droplets of the dispersed phase float or sink under the action of centrifugal force, and the more stable the emulsion, the results are shown in Table 1.

Table 1 Quality evaluation results of saponin II emulsion

Note: “+” means yes; “-” means none; compared with other groups: *P<0.05

Experimental results: According to the types of excipients used in the formulation of the present invention, the quality of the saponin II emulsion can be prepared, and the appearance of the emulsion has no delamination, flocculation, sticking, etc. Among them, the experimental group 6 and other experimental groups The particle size and Ke value of the emulsion were significantly reduced (P<0.05), indicating that the most suitable excipients for the preparation of the saponin II emulsion were: soybean oil as the oil phase, soybean phospholipid as the emulsifier, and oleic acid as the stabilizer. Glycerin is an osmotic pressure regulator.

Experimental Example 2 Effect of Excipient Amount on the Quality of Saponin II Emulsion

Seven experimental groups were set up in this experiment. The saponins II, soybean oil, and soybean phospholipids (fixed saponin II weight 0.6 g) were weighed according to the weight ratios shown in Table 2, dissolved in ethanol, and ethanol was removed by rotary evaporation under reduced pressure. II.. Oleic acid: glycerol is added with oleic acid and glycerol at a high speed of 1:0.5:1, and water is added to the emulsion. The concentration of saponin II is 0.3 mg/mL, which is high at 10,000 rpm. Speed shearing for 5 min, high pressure homogenization 4 times under 800 bar pressure, autoclaving. The appearance was observed for delamination, flocculation, and sticking, and the particle size was measured to determine Ke. The results are shown in Table 2.

Table 2 Quality evaluation results of saponin II emulsion

Experimental results: The saponin II: soybean oil: soybean phospholipid weight ratio in the range of 1:1 ~ 350: 0.5 ~ 40, can produce better quality emulsion; among them, experimental group 6 compared with other experimental groups The particle size and Ke value of the emulsion were significantly reduced (P<0.05), indicating that the optimal dosage of the excipients in the saponin II emulsion was: saponin II: soybean oil: soybean phospholipid = 1:300:20.

Experimental Example 3 Pharmacodynamic experiment of the saponin II emulsion of the present invention

1. Experimental materials, reagents and instruments

1.1. Test drug: The group of the saponin II fat emulsion solution (A, B, C, D, E, F, G) prepared according to Example 1, the saponin II 10% DMSO-salt group.

1.2, tool drugs: cyclophosphamide.

1.3. Experimental animal KM-mouse: 18.5-22.5 g.

1.4, experimental equipment: automatic blood cell analyzer; BS-600L electronic balance: specifications: 600g / 0.1g, Shanghai Yousheng Weighing Apparatus Co., Ltd.

2, statistical methods

Statistical analysis was performed using SPSS 17.0 software. Data in mean ± standard deviation

One-way ANOVA was used between the groups. The LSD test was performed between the groups with variance, and the Tamhane's T2 test was performed for those with irregular variance.

3. Experimental methods

3.1, experimental animal grouping and model preparation

All animals were fed ad libitum for 1 week and were randomly divided into: blank group; model group; different prescriptions of saponin II emulsion group (A, B, C, D, E, F, G), formulated into 2.5 mg·kg -1 suspension, prepared before use; saponin II group: saponin II powder, dissolved in 10% DMSO-physiological saline, formulated into 2.5mg·kg-1 suspension, prepared before use. On the first day of the experiment, except for the blank group, the other groups of mice were intraperitoneally injected with cyclophosphamide physiological saline solution at a dose of 50 mg·kg -1 for 3 consecutive days. The blank group of mice was injected with an equal volume of normal saline in the tail vein.

3.2, administration

Each experimental group was given the corresponding drug at the dose and iv from the first day of the experiment. The blank group and the model group were injected with the same volume of normal saline in the tail vein for 7 consecutive days.

3.3, specimen collection

On the 8th day of the experiment, blood was taken from the eye of each experimental group and collected with a 0.5 ml EP tube containing EDTA anticoagulant.

3.4. Test indicators and methods

(1) Peripheral blood test: Peripheral blood leukocytes (WBC), neutrophils (NEUT) red blood cells (RBC), platelets (PLT), and hemoglobin (HGB) were counted in each experimental group by an automatic blood cell counter.

(2) Bone marrow hematopoietic stem cell count (based on bone marrow cell CD34+ antigen expression) The right femur bone marrow cells were washed out with PBS buffer containing 0.2% bovine serum albumin, and 106 cells were removed and centrifuged. 30 μL of normal mouse serum was added to block the non-specific binding site, 10 μL of FITC-labeled rat anti-mouse CD34+ antibody was added, 10 μL of the corresponding control antibody was added to the control tube, and the reaction was incubated at 4° C. for 30 min in the dark. 2 mL of red blood cell lysate was added for 5 min, the cells were washed twice, and PI staining solution with a final concentration of 3 μg/mL was added, and the expression of CD34+ antigen in bone marrow cells was detected by flow cytometry.

4. Experimental results

4.1. Comparison of major blood cell counts in peripheral blood

Table 3 Number of peripheral blood cells in each experimental group

Note: Compared with the model group, *P<0.05, **P<0.01; Note: Compared with the saponin II group, ^△ P<0.05, ^△△ P<0.01.

As can be seen from Table 3, compared with the model group, the number of WBC, RBC, and PLT in the peripheral blood of the saponin II emulsion group of the present invention was significantly increased (P<0.05), and there was no significant difference in the saponin II group; Compared with the group of saponins II, the number of WBC, RBC and PLT in the peripheral blood of mice in the saponin II emulsion group of the present invention was significantly increased (P<0.05).

Table 4 Number of NEUT and HGB in peripheral blood of mice in each experimental group

Note: Compared with the model group, *P<0.05, **P<0.01; compared with the saponin II group, ^△ P<0.05, ^△△ P<0.01.

As can be seen from Table 4, compared with the model group, the NEUT and HGB numbers in the peripheral blood of the saponin II emulsion group of the present invention were significantly increased (P<0.05), and there was no significant difference in the saponin II group; Compared with the saponin II group, the NEUT and HGB levels in the peripheral blood of the mice in the saponin II emulsion group of the present invention were significantly increased (P<0.05).

4.2, comparison of bone marrow hematopoietic stem cell count

As can be seen from Fig. 1, compared with the model group, the number of hematopoietic stem cells in the saponin II emulsion group of the present invention was significantly increased (P<0.05), and there was no significant difference in the saponin II group; In comparison, the number of hematopoietic stem cells in the saponin II emulsion group of the present invention was significantly increased (P<0.05).

The above experimental results show that the preparation of the saponin II as an emulsion according to the formulation of the present invention can significantly improve the blood cell level of the drug, and can effectively prevent bone marrow suppression.

Claims

An earthworm saponin II emulsion, which is prepared by preparing a raw material having the following weight ratio: 1 part of saponin II, 1-350 parts of oil phase, 0.5-40 parts of emulsifier.
The emulsion according to claim 1, which is prepared by containing a raw material of the following weight ratio: 1 part of saponin II, 300 parts of an oil phase, and 20 parts of an emulsifier.
The emulsion according to claim 1 or 2, wherein the oil phase is one or more selected from the group consisting of soybean oil, medium chain triglyceride, fish oil, olive oil, ethyl oleate, and castor oil. a mixture; the emulsifier is selected from one or both of egg yolk lecithin and soybean phospholipid.
The emulsion according to any one of claims 1 to 3, which further comprises 0.5 to 5 parts of a stabilizer and 1 to 10 parts of an isotonicity adjusting agent.
The emulsion according to claim 4, wherein said stabilizer is oleic acid; and said isotonicity adjusting agent is glycerin.
The emulsion according to any one of claims 1 to 5, which is prepared from the following raw materials by weight ratio: 1 part of saponin II, 300 parts of soybean oil, 20 parts of soybean phospholipid, 0.5 parts of oleic acid and 1 part of glycerin were added to the concentration of saponin II of 0.3 mg/mL.
A method for preparing an emulsion according to any one of claims 1 to 6, characterized in that it comprises the following steps:

a, weigh each weight ratio of saponin II, oil phase and emulsifier, dissolved in organic solvent, mixed solution for use;

b, removing the organic solvent in the mixed solution of step a, and then adding a stabilizer, glycerin and water;

c, shear, homogenize, sterilize, that is.
The preparation method according to claim 7, wherein the organic solvent is ethanol; the shearing condition is: shearing at 10,000 rpm for 5 minutes; and the homogenizing condition is: homogenizing at a pressure of 800 bar. Times.
Use of an emulsion according to any of claims 1-6 for the manufacture of a medicament for the treatment and/or prevention of myelosuppression.
Use of the emulsion according to any one of claims 1 to 6 for the preparation of a medicament for increasing the amount of blood cells and hemoglobin.
A method of treating and/or preventing myelosuppression, characterized by using the emulsion of any one of claims 1-6.
A method for increasing the number of blood cells and hemoglobin, characterized by using the emulsion according to any one of claims 1 to 6.