CN1116028C - Nanometer-size realgar and its preparation - Google Patents

Abstract

The invention introduces the nanotechnology into the preparation of the traditional Chinese medicine realgar, that is, the realgar is made into nanoscale (1-100 nanometers) fine powder, thereby endowing the realgar with higher drug efficacy and antitumor effect. Since realgar is insoluble in water, it is generally taken by grinding it into powder and taking it as medicine. Studies have shown that the pharmacological effects of traditional Chinese medicine cannot be solely attributed to the unique chemical composition of the drug, but are also closely related to the physical state of the drug. Therefore, nano-realgar has been proved by drug efficacy experiments to significantly improve drug efficacy, and nano-realgar is expected to become a new class of antitumor drug with high efficiency and low toxicity. The method for realizing the nanometer realgar of the present invention is: (1) a temperature-controllable vacuum or inert atmosphere high-energy ball milling method; (2) a supersonic airflow pulverization method.

Description

Nano realgar and its preparation method

technical field

What the present invention relates to is traditional Chinese medicine and preparation method thereof. Introduce nanotechnology to the preparation of realgar, a traditional Chinese medicine, that is, make realgar into nano-scale fine powder, so as to endow realgar with higher efficacy. Nanotechnology refers to the research and application on the material scale of 0.1-100 nanometers (nanometer, nanometer for short).

Background technique

Realgar, a mineral traditional Chinese medicine, is mainly composed of arsenic disulfide (As ₂ S ₂ ). The functions and indications of realgar are to detoxify and kill insects, dry dampness and expel phlegm, cut off malaria, and be used for carbuncle and furuncle, snake and insect bites, abdominal pain caused by parasites, epilepsy, malaria, etc. Modern pharmacological studies have shown that realgar has anti-pathogenic microorganisms and anti-tumor effects.

Realgar is insoluble in water, and it is generally taken as medicine by grinding it into powder. But there is no report that realgar is made into nanoscale powder.

In the past, it was generally believed that the material basis of traditional Chinese medicine for disease prevention and treatment came from biologically active components or active chemical components. However, the pharmacological effects of traditional Chinese medicine cannot be solely attributed to the unique chemical composition of the medicine, and may also be closely related to the physical state of the medicine. Due to the lack of advanced processing methods in traditional Chinese medicine processing methods, the bioavailability of many medicines is low, and it is difficult to fully exert their efficacy.

Contents of the invention

The invention aims at making the realgar into powder with nanometer particle size (1-100nm) to improve the drug effect of the traditional Chinese medicine realgar and the higher anti-tumor effect of the realgar particles in the nanometer state.

The present invention uses two kinds of methods for preparing nano-realgar, (1) 0.1-2.0 kilograms of raw material realgar powder is placed in a vacuum or inert gas atmosphere ball mill jar equipped with a cryogenic jacket, and 1.0-20 kilograms of milling jars are simultaneously loaded. Balls, keep the ratio of balls and realgar powder in the range of 15:1 to 5:1, control the speed and time of the high-energy ball mill so that it is in the range of 200 to 400 RPM (rev/min) and 2 to 60 hours to obtain the average particle size Realgar powder smaller than 100 nanometers. The ball mill adopts a double-layer jacket structure, and the jacket is equipped with a liquid nitrogen injection port and a temperature detection device. The operating temperature is controlled by adjusting the amount of liquid nitrogen injected into the double-layer jacket, and the control range is -50°C to 100°C. The operating atmosphere in the high-energy ball mill is vacuum or inert gas; (2) Supersonic jet milling method: use a commercially available jet mill, the operating atmosphere in the machine is inert gas, and the temperature can be controlled within the range of -50°C to 100°C. The inert gases used in the above two methods are nitrogen and carbon dioxide. Observed by an electron microscope, the particle diameters of the realgar powder prepared by these two methods all reach the nanometer level, that is, 1 to 100 nanometers.

Detailed ways

Take nano powder realgar and common realgar powder for drug effect comparison experiment. In the following two experiments, several realgar powder samples with different particle sizes were prepared from realgar, with the purpose of studying the influence of the particle size of the powder, especially the nanoscale, on its pharmacological effects. Experiment (1) Study on Inhibition of Mouse Sarcoma S ₁₈₀ Tumor Cells in Vitro

Materials for this experiment: realgar was purchased from the pharmacy of Hubei Institute of Traditional Chinese Medicine, and Kunming mice (about 20 g) and S ₁₈₀ tumor-bearing mice were purchased from the Experimental Animal Center of Tongji Medical University. The experimental method:

1. Obtained three kinds of realgar powder samples with different particle sizes by the above-mentioned preparation method, and its average particle size is respectively 500nm (No. I sample), 200nm (No. II sample) and <100nm (No. III sample).

2. prepare the stable suspension of different realgar samples with 1% sodium carboxymethylcellulose aqueous solution as suspending agent, and press three different doses of 1.25g/Kg, 0.75g/Kg, 0.25g/Kg body weight respectively to Kunming kind The mice were given intraperitoneal injection, and the number of experimental animals in each group was 5, a total of nine groups, and the adverse reactions and death of the mice at different times were investigated.

3. Extract S ₁₈₀ ascites tumor cells from the peritoneal cavity of tumor-bearing mice, and dilute them to 6×10 ⁷ cell suspension per ml with sterile saline. Add different doses of realgar suspension to the cell suspension in the test tube, incubate at 37°C, and shake it from time to time. The morphological changes and death of tumor cells at different times were observed by trypan blue staining. Results and discussion:

No animal death or obvious adverse reaction was observed in the three different dose groups of the acute toxicity No. I sample within the investigated time (1-24 hours). No. II and No. III sample results are similar, and the 0.25g/Kg dose group has no animal death in the time (1-24 hours) investigated, and there is no obvious adverse reaction; 20% of the animals died; all animals in the 1.25g/Kg dose group died 4 hours after administration. The result shows that realgar samples of different particle sizes, its acute toxicity: No. III sample (<100nm) ≈ No. II sample (200nm) >> No. I sample (500nm) antitumor effect: 25mg/ml dosage group, three kinds of realgar and cell After acting for 10 minutes, microscopic examination showed different degrees of cell morphology changes, such as cell membrane wrinkling, but no cell death was seen. Table 1 shows the toxic effect of realgar powder with different particle sizes on _S180 tumor cells after 2 hours.

The poisonous effect ( ₂ hours) dosage 25mg/ml 20mg/ml 15mg/ml 10mg/ml 5mg/mlI sample (500nm) 90% 89% 60% 8% 12 %II sample (200nm) 100% 95% 92% 90% 87%III sample (<100nm) 100% 95% 93% 93% 70%

Illustrate (take action 2 hours as example), the realgar samples of different particle diameters, to S ₁₈₀ tumor cell poisonous action intensity order is: No. III sample (<100nm) ≈ No. II sample (200nm) >> No. I sample (500nm) The above results indicated that the antitumor effect of realgar, a mineral traditional Chinese medicine, had an obvious size effect. Experiment (2) Size Effect of Realgar Induced Apoptosis of Human Umbilical Vein Endothelial Cell Line ECV-304

Tumor angiogenesis is the pathological basis of tumor growth and metastasis. Tumor angiogenesis inhibitors (tumor angiogenesis inhibitor, TAI) is a class of drugs that can destroy or inhibit angiogenesis, and effectively prevent tumor growth and metastasis.

In this experiment, the effects of realgar granules with different particle sizes on the survival rate and apoptosis of ECV-304 cells were studied. Methods Realgar granules with particle diameters of <100nm, 150nm, 200nm, and 500nm were co-cultured with cells, and the concentration of realgar was 5×10 ^-8 mol/L, 10×10 ^-8 mol/L, 20× Cell viability under the condition of 10 ^-8 mol/L: Fluorescence microscope observation, agarose gel electrophoresis and flow cytometry to qualitatively and quantitatively detect males with different particle sizes at a concentration of 20×10 ^-8 mol/L induced apoptosis. Results: (1) Under the conditions of 2, 4, 6, and 8 hours of treatment respectively, realgar <100nm and 150nm could significantly reduce the cell survival rate at the three concentrations studied, and showed time effects and concentration effects, while 200nm and 500nm realgar had no obvious effect on the cell survival rate under the same conditions. See attached drawings A, B, C: "Effects of realgar on the survival rate of ECV-304 endothelial cells", where the concentration of realgar in A is 5×10 ^{- 8} mol/L, B is 10×10 ^-8 mol/L, C is 20×10 ^-8 mol/L, the ordinate is the average percent survival rate, and the abscissa is the culture time (hours); each curve indicates the control group, ■ indicates the <100nm realgar group, △ indicates the 150nm realgar group, × indicates the 200nm realgar group, and □ indicates the ^500nm realgar group; After the cells were treated at the concentration for 8 hours, the fluorescent dye was stained with pyridine orange, and the cell morphology was observed under the fluorescence microscope. The results showed that the cells showed obvious apoptosis when the particle size was less than 100nm and 150nm: the cell membrane foamed and shrunk, and the cell volume Smaller, apoptotic bodies formed, chromatin tightened, and the other two particle sizes of males treated cells did not show obvious apoptosis; DNA gel electrophoresis of cells treated with <100nm and 150nm realgar showed typical apoptosis Apoptotic features: a ladder-like pattern formed by regular DNA breaks; flow cytometry detected the treated cells, and there was a sub-diploid apoptotic peak before the G1 phase of the cell cycle, and the statistical results of the apoptotic peak area showed that the corresponding granule diameter <100nm-500nm, the apoptosis rates were: 68.15%, 49.62%, 7.51%, 5.21%.

This study shows that realgar with a particle size of <100nm and 150nm can reduce the survival rate of endothelial cells by inducing apoptosis. This result suggests that realgar with small particle size is expected to become a new type of tumor vascular with high efficiency and low toxicity. Generate inhibitors.

Nanotechnology is a high-tech that manipulates atoms and molecules within the spatial scale of 0.1-100nm, processes materials, and manufactures products with specific functions. technology". When the particle size enters the nanoscale, due to the quantum size effect and surface effect, nanomaterials present many novel physical, chemical and biological properties. The invention relates to the introduction of nanotechnology into the preparation of realgar, that is, making realgar into nano-scale ultrafine powder, so as to endow realgar with higher drug efficacy.

Figures A, B, C: Effects of realgar on the survival rate of endothelial cells ECV-304, where the concentration of realgar in A is 5×10 ^-8 mol/L, B is 10×10 ^-8 mol/L, and C is 20× 10 ^-8 mol/L, the ordinate is the average percent survival rate, and the abscissa is the culture time (hours); each curve ◆ indicates the control group, ■ indicates the 100nm realgar group, △ indicates the 150nm realgar group, and × indicates the 200nm realgar group, □ indicates the 500nm realgar group.

Example: Take 1.5 kg of realgar and 15 kg of balls and place them in a vacuum or inert gas atmosphere ball mill jar equipped with a cryogenic jacket. The flow rate of liquid nitrogen is 50 ml/min, the temperature is controlled at 0°C, and the speed of the high-energy ball mill is Be 300RPM (rev/min), run 20 hours, promptly obtain the realgar powder that average particle diameter is less than 100 nanometers, and obtain the proof of electron microscope observation.

Claims (3)

1. the Powdered Realgar as Chinese medicine is characterized in that, the particle diameter of Powdered Realgar is 1～100 nanometer.

2. method for preparing Powdered Realgar as claimed in claim 1, it is characterized in that: this method is that the Realgar material powder is placed the high energy ball mill jar, make ball powder ratio remain on 15: 1～5: 1 ratio, be filled with vacuum or inert gas atmosphere in jar, the rotating speed and the time of control high energy ball mill, make it in 200～400RPM and 2～60 hours scopes, the control temperature, span of control is-50 ℃～100 ℃, and temperature is to control by the amount that adjusting is injected into the double-layer sleeve liquid nitrogen of high energy ball mill.

3. method for preparing pulverous Realgar as claimed in claim 1, this method is that the Realgar material powder is placed in the jet mill, in inert gas atmosphere, pulverize, it is characterized in that, the control temperature, span of control is-50 ℃～100 ℃, and temperature is to control by the amount of liquid nitrogen that adjusting is injected in the jet mill.

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