CN116807996A - Beta-acid loaded chitosan nanomaterial and preparation method and application thereof - Google Patents
Beta-acid loaded chitosan nanomaterial and preparation method and application thereof Download PDFInfo
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- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
- A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
- A61K9/51—Nanocapsules; Nanoparticles
- A61K9/5192—Processes
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- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
- A61K31/19—Carboxylic acids, e.g. valproic acid
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- A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
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Abstract
The invention provides a beta-acid loaded chitosan nanomaterial and a preparation method and application thereof, and relates to the technical field of preparation of beta-acid loaded chitosan nanomaterial by an ion gel method, wherein chitosan is used as a reactant, sodium tripolyphosphate is used as a cross-linking agent, and beta-acid is used as an active ingredient, so that beta-acid is simple to deliver, the reaction process is concise, safe, stable and controllable, the requirements on equipment and environment are low, and the environment is protected; and the prepared chitosan nano-material loaded with the beta-acid has high drug loading capacity and high coating rate, the drug targeting property and the stability are greatly improved, and the prepared chitosan nano-material loaded with the beta-acid has good antibacterial and anticancer effects.
Description
Technical Field
The invention belongs to the technical field of chitosan nano materials, and particularly relates to a beta-acid loaded chitosan nano material, and a preparation method and application thereof.
Background
Along with the continuous acceleration of the life rhythm of people, unhealthy life habits can lead to obvious increase of occurrence probability of diseases such as cancers, inflammations and the like, so that more, alternative and effective medicaments need to be developed for ensuring the safety and the health of human beings, in the prior art, more and more functional materials are prepared by the rapid development of nano technology, so that the application of the nano-technology in various fields is expanded, the life and the health of the human beings are greatly influenced, and the use interest of the nano-particles by people is exponentially increased; the invention provides a novel nano material for preparing anticancer drugs.
Disclosure of Invention
In view of the above, the present invention provides a β -acid loaded chitosan nanomaterial for preparing anticancer drugs.
It is also necessary to provide a method for preparing the beta-acid loaded chitosan nanomaterial.
It is also necessary to provide an application of the beta-acid loaded chitosan nanomaterial.
The technical scheme adopted for solving the technical problems is as follows:
a method for preparing a beta-acid loaded chitosan nanomaterial comprises the following steps,
s1: dissolving chitosan in acetic acid solution to obtain chitosan solution, and dissolving beta-acid in absolute ethyl alcohol to obtain beta-acid solution;
s2: mixing the beta-acid solution with the chitosan solution to obtain a mixed solution;
s3: dropwise adding a sodium tripolyphosphate solution into the mixed solution to perform ionic gel method reaction for a preset time to generate a chitosan nanoparticle solution containing beta-acid;
s4: and centrifuging and freeze-drying the chitosan nanoparticle solution containing the loaded beta-acid to obtain the chitosan nanoparticle loaded with the beta-acid.
Preferably, in the step S1, the concentration of the chitosan solution is 1mg/mL-10mg/mL, and the concentration of the beta-acid solution is 1mg/mL-30mg/mL.
Preferably, in the step S2, the volume ratio of the beta-acid solution to the chitosan solution is 1:0.25-1.5.
Preferably, in the step S2, when the β -acid solution is mixed with the chitosan solution, the mixed solution is obtained by stirring for 10min to 30min at normal temperature.
Preferably, in the step S3, the concentration of the sodium tripolyphosphate solution is 1mg/mL-10mg/mL.
Preferably, in the step S3, the mass ratio of the sodium tripolyphosphate solution to the mixed solution is 1:0.5-2.
Preferably, in the step S3, a sodium tripolyphosphate solution is added dropwise to the mixed solution at normal temperature, and the reaction is performed for a predetermined time of 1-2 hours.
The beta-acid-loaded chitosan nanomaterial prepared by the preparation method of the beta-acid-loaded chitosan nanomaterial.
The application of the beta-acid loaded chitosan nanomaterial in preparing antibacterial or anticancer drugs.
The application of the beta-acid loaded chitosan nanomaterial in preparing medicines for inhibiting staphylococcus aureus or escherichia coli or resisting colon cancer is provided.
Compared with the prior art, the invention has the beneficial effects that:
according to the beta-acid-loaded chitosan nanomaterial, the preparation method and the application thereof, provided by the invention, the beta-acid-loaded chitosan nanomaterial is prepared by an ion gel method, chitosan is used as a reactant, sodium tripolyphosphate is used as a cross-linking agent, and beta-acid is used as an active ingredient, so that the beta-acid is simple to deliver, the reaction process is concise, safe, stable and controllable, the requirements on equipment and environment are low, and the environment is protected; and the chitosan nanoparticle with proper size, potential and drug loading capacity and loaded with beta-acid is prepared by an ion gel method.
The prepared beta-acid loaded chitosan nanomaterial has high drug loading rate and coating rate, greatly improves drug targeting property and stability, and has great effect in bacteriostasis and anticancer.
Drawings
Fig. 1 is a scanning electron microscope diagram of a second embodiment.
FIG. 2 is an infrared spectrum of a second embodiment.
Fig. 3 is an XRD pattern of example two.
Fig. 4 is an in vitro release profile of β -acids in nanoparticles for example one, example two, example three, example four.
In the figure: in fig. 1: blank chitosan nanoparticles: (a) 1 μm, (b) 500 nm; example two: (c) 1 μm, (d) 500nm scanning electron microscope image;
in fig. 2: (a) chitosan, (b) sodium tripolyphosphate, (c) blank chitosan nanoparticles, (d) example two;
in fig. 3: (a) chitosan, (b) sodium tripolyphosphate, (c) beta-acid, (d) blank chitosan nanoparticles, (e) example two;
in fig. 4: (a) instance one, (b) instance two, (c) instance three, and (d) instance four.
Detailed Description
The technical scheme and technical effects of the embodiments of the present invention are further described in detail below with reference to the accompanying drawings.
A method for preparing a beta-acid loaded chitosan nanomaterial comprises the following steps,
s1: dissolving chitosan in acetic acid solution to obtain chitosan solution, and dissolving beta-acid in absolute ethyl alcohol to obtain beta-acid solution;
s2: mixing the beta-acid solution with the chitosan solution to obtain a mixed solution;
s3: dropwise adding a sodium tripolyphosphate solution into the mixed solution to perform ionic gel method reaction for a preset time to generate a chitosan nanoparticle solution containing beta-acid;
s4: and centrifuging and freeze-drying the chitosan nanoparticle solution containing the loaded beta-acid to obtain the chitosan nanoparticle loaded with the beta-acid.
Compared with the prior art, the invention has the beneficial effects that:
according to the beta-acid-loaded chitosan nanomaterial, the preparation method and the application thereof, provided by the invention, the beta-acid-loaded chitosan nanomaterial is prepared by an ion gel method, chitosan is used as a reactant, sodium tripolyphosphate is used as a cross-linking agent, and beta-acid is used as an active ingredient, so that the beta-acid is simple to deliver, the reaction process is concise, safe, stable and controllable, the requirements on equipment and environment are low, and the environment is protected; and the chitosan nanoparticle with proper size, potential and drug loading capacity and loaded with beta-acid is prepared by an ion gel method.
The prepared beta-acid loaded chitosan nanomaterial has high drug loading rate and coating rate, greatly improves drug targeting property and stability, and has great effect in bacteriostasis and anticancer.
Further, in the step S1, the concentration of the acetic acid is 0.1-1% (v/v), so that chitosan can be well dissolved, and the solution is prevented from being too strong in acidity, so that the structure of the chitosan is damaged.
Further, in the step S1, the concentration of the chitosan solution is 1mg/mL-10mg/mL, the concentration of the beta-acid solution is 1mg/mL-30mg/mL, and the prepared beta-acid-loaded chitosan nanomaterial in the concentration range has uniform particle size and stable property.
Further, in the step S2, the volume ratio of the beta-acid solution to the chitosan solution is 1:0.25-1.5, so that the drug loading rate of the prepared drug-loaded nano-particles is gradually increased, and finally the platform phase is reached.
Further, in the step S2, the volume ratio of the beta-acid solution to the chitosan solution is 1:0.25-1.
Further, in the step S2, when the β -acid solution is mixed with the chitosan solution, the mixed solution is obtained by stirring for 10min to 30min at normal temperature.
Further, in the step S3, the concentration of the sodium tripolyphosphate solution is 1mg/mL-10mg/mL, so that the reaction is moderate and the time is saved; the concentration is prevented from being too high, the reaction is too fast, the concentration is too low, the reaction speed is very slow, and the time is wasted.
Further, in the step S3, the mass ratio of the sodium tripolyphosphate solution to the mixed solution is 1:0.5-2.
Further, in the step S3, the mass ratio of the sodium tripolyphosphate solution to the mixed solution is 1:2, so that the prepared chitosan nanomaterial loaded with the beta-acid is uniform and stable in particle size.
Further, in the step S3, a sodium tripolyphosphate solution is dropwise added into the mixed solution at normal temperature, and the reaction preset time is 1-2h.
The beta-acid-loaded chitosan nanomaterial prepared by the preparation method of the beta-acid-loaded chitosan nanomaterial.
The application of the beta-acid loaded chitosan nanomaterial in preparing antibacterial or anticancer drugs.
The application of the beta-acid loaded chitosan nanomaterial in preparing medicines for inhibiting staphylococcus aureus or escherichia coli or resisting colon cancer is provided.
Example 1
600mg of chitosan was dissolved in 200ml of acetic acid solution (1% v/v), and then undissolved chitosan was removed through a 1 μm microporous filter membrane to prevent undissolved chitosan particles from affecting the next reaction, resulting in 3mg/ml of chitosan solution. The beta-acid is dissolved in absolute ethanol to obtain beta-acid solution with the concentration of 15mg/ml. The beta-acid solution (15 mg) was then mixed with 200mL of chitosan solution. Then, sodium tripolyphosphate solution (2 mg/ml,15 ml) was slowly dropped, stirred at 800rpm, 25℃for reaction for 45 minutes, the formed nanoparticles were centrifuged at 3000 Xg at 4℃for 10 minutes, washed 3 times with deionized water, the centrifuged nanoparticles were put into a 1.5ml EP tube, frozen in a refrigerator at-80℃and the EP tube was put into a freeze-dryer (Croos-80, telstar) for 24 hours at a temperature of-85℃and 0.02kPa to obtain beta-acid-loaded chitosan nanoparticles.
Example two
300mg of chitosan was dissolved in 50ml of acetic acid solution (1% v/v), and then undissolved chitosan was removed through a 1 μm microporous filter membrane, to obtain a chitosan solution of 6 mg/ml. Beta-acid was dissolved in absolute ethanol at a concentration of 10mg/ml. The beta-acid solution (30 mg) was then mixed with chitosan solution (50 mL). Then, sodium tripolyphosphate solution (1 mg/ml,15 ml) was slowly dropped, stirred at 800rpm, 25℃for reaction for 60 minutes, the formed nanoparticles were centrifuged at 3000 Xg at 4℃for 10 minutes, washed 3 times with deionized water, the centrifuged nanoparticles were put into a 1.5ml EP tube, frozen in a refrigerator at-80℃and the EP tube was put into a freeze-dryer (Croos-80, telstar) for 24 hours at a temperature of-85℃and 0.02kPa to obtain beta-acid-loaded chitosan nanoparticles.
Example III
Taking 30mL of water in a 50mL beaker, sequentially adding 1mL of acetic acid and 150mg of chitosan, stirring at 25 ℃ until the mixture is completely dissolved, and dissolving beta-acid in absolute ethanol with the concentration of 30mg/mL. The beta-acid solution (60 mg) was then mixed with chitosan solution (30 mL). Then, sodium tripolyphosphate solution (4 mg/ml,15 ml) was slowly dropped, stirred at 800rpm, 25℃for reaction for 45 minutes, the formed nanoparticles were centrifuged at 3000 Xg at 4℃for 10 minutes, washed 3 times with deionized water, the centrifuged nanoparticles were put into a 1.5ml EP tube, frozen in a refrigerator at-80℃and the EP tube was put into a freeze-dryer (Croos-80, telstar) for 24 hours at a temperature of-85℃and 0.02kPa to obtain beta-acid-loaded chitosan nanoparticles.
Example IV
900mg of chitosan was dissolved in 200ml of acetic acid solution (1% v/v), and then undissolved chitosan was removed through a 1 μm microporous filter membrane, to obtain 4.5mg/ml of chitosan solution. Beta-acid was dissolved in absolute ethanol at a concentration of 20mg/ml. The beta-acid solution (90 mg) was then mixed with chitosan solution (200 mL). Then, sodium tripolyphosphate solution (10 mg/ml,15 ml) was slowly dropped, stirred at 800rpm, 25℃for reaction for 45 minutes, the formed nanoparticles were centrifuged at 3000 Xg at 4℃for 10 minutes, washed 3 times with deionized water, the centrifuged nanoparticles were put into a 1.5ml EP tube, frozen in a refrigerator at-80℃and the EP tube was put into a freeze-dryer (Croos-80, telstar) for 24 hours at a temperature of-85℃and 0.02kPa to obtain beta-acid-loaded chitosan nanoparticles.
The beta-acid-loaded chitosan nanoparticles obtained in the first to fourth examples were subjected to electron microscope scanning, infrared spectroscopy, X-ray diffraction, drug loading and encapsulation efficiency, release, bacteriostasis and anticancer performance tests, and the results were as follows:
1. scanning electron microscope
The beta-acid-loaded chitosan nanoparticle obtained in the second example was subjected to scanning electron microscopy, and the particle was spherical as shown in fig. 1, and the nanoparticle without beta-acid was observed to be in an aggregated state, but had a smooth surface. The apparent size of the nanoparticles at the same scale becomes significantly larger after the addition of the β -acid, probably due to the aggregation of the β -acid molecules to form a fully covered surface. The surface of the β -acids seems to be more "sticky" than the blank nanoparticles, indicating that the addition of β -acids to the nanoparticles during their formation may affect the assembly process of the nanoparticles.
2. Infrared spectrum
The beta-acid loaded chitosan nanoparticle obtained in the second example was subjected to infrared spectrum test, and after free chitosan and sodium tripolyphosphate formed nanoparticles by ion gel technology, the characteristic peak in the infrared spectrum was found to be changed, as shown in fig. 2. When the beta-acid is loaded in the nanoparticle, the-OH in the beta-acid interacts with the-OH in the nanoparticle, so that the-OH absorption peak of the drug-loaded nanoparticle appears at 3397cm -1 Where it is located. Intermolecular hydrogen bonds (c=o and C-O bonds) are formed between the β -acid and the nanoparticle, and the absorption peak is found to be 1644cm earlier than that of the blank nanoparticle in the spectrum -1 And 1073cm -1 The absorption peak at the location shifted to 1630cm -1 And 1088cm -1 Where (shown as d in fig. 2). These results further confirm the successful preparation of β -acid loaded nanoparticles.
3.X-ray diffraction
When the beta-acid-loaded chitosan nanoparticle obtained in example two was subjected to XRD test, it was clearly observed that the chitosan powder had a significant CHs diffraction peak at a diffraction angle of 2θ=20° as shown in fig. 3, and exhibited high intensity reflection. The pure TPP powder has absorption peaks around different diffraction angles of 2θ=20 °,25 °, 30 °, 33 °, 55 °, etc. fig. 3c. After the ionic crosslinking reaction of chitosan and TPP, the positions of diffraction peaks of the formed nanoparticles were shifted (2θ=22°), and the diffraction peaks exhibited a tendency to widen. Furthermore, the X-ray diffraction pattern of pure β -acid shows that there are several characteristic peaks near 2θ=8°,9 °,16 ° and 18 °, indicating that it has a certain crystallinity. When the β -acids are entrapped in the nanoparticles, these crystallization peaks disappear, possibly due to the formation of an amorphous state. The XRD diffractogram of the drug-loaded nanoparticle showed a new diffraction peak around 2θ=12°, and the diffraction peak appeared amorphous.
As can be seen from electron microscope scanning, infrared spectrum and X-ray diffraction, the beta-acid loaded chitosan nanoparticle is prepared by the method.
4. Particle size and potential
The average particle size, polydispersity index (PDI) and Zeta potential of the β -acid loaded chitosan nanoparticles prepared in examples one to four were measured on a Zetasizer Nano (Malvern, UK). The sample was uniformly dispersed in ultrapure water by ultrasonic wave before measurement, and the test results are shown in table 1:
TABLE 1
From the data obtained, it can be seen that the average diameter of the drug-loaded nanoparticles is in the range of 240-260nm, allowing access to the blood. The polydispersity index (PDI) of the beta-acid drug-loaded nano-particles with different contents is 0.27,0.37,0.44 and 0.94 respectively, and the dispersing effect is good. In addition, the zeta potential of the nano-particles with different drug loading amounts is 14.47,14.80,16.17 and 16.20mV respectively, so that the nano-particles can be contacted with cell membranes, and can enter blood more easily.
5. Drug loading and encapsulation efficiency
The encapsulation efficiency of the beta-acid in the chitosan nanoparticle is determined by adopting an ultraviolet-visible spectrum method. The sample of β -acid-loaded chitosan nanoparticle prepared in examples one to four (5 mg) was suspended in 30mL of 10% ethanol, centrifuged at 4000 rpm for 10 minutes, 5mL of absolute ethanol solution was added after discarding the supernatant, and after sonicating for 1 hour, the absorbance value of the supernatant was measured after centrifuging the mixture at 4000 rpm for 10 minutes. With the standard curve of beta-acids in ethanol (R 2 =0.992) as a reference, the absorbance of the supernatant was measured at 365 nm. The chitosan nanomaterial treatment was similar to that described above and was used for the blank. Three determinations were made for each sample. The Encapsulation Efficiency (EE) and the loading energy (LG) of the β -acid are calculated from the following formulas:
the test results are shown in Table 2.
TABLE 2
As the β -acid content increases, the EE and LC of the drug-loaded nanoparticles gradually increase. When the mass ratio of chitosan to beta-acid is 1:1.5, the maximum EE (55.94%) and LC (16.91%) are obtained, so that the beta-acid has good drug loading rate and highest encapsulation efficiency.
6. Release of
The particles were incubated in different pH buffer solutions (ph=7.4, 6.6 and 1.2, mimicking normal human tissue, tumor microenvironment and gastric fluid environment, respectively) at 37 ℃. Experiments show that under three different pH conditions, the beta-acid-loaded chitosan nanoparticles prepared in examples one to four are released rapidly (burst release) and then released slowly, and the beta-acid-loaded chitosan nanoparticles prepared in examples one to four have small release amount in gastric juice environment, and most of the beta-acid-loaded chitosan nanoparticles can enter colorectal tissues for release, so that targeting property is good and stable.
7. Antibacterial agent
The staphylococcus aureus (ATCC 25923) and escherichia coli (ATCC 25922) strains are taken, the nutrient broth is added under the aseptic condition, and the culture is expanded and carried out at 37 ℃ for 24 hours. A representative colony was selected and dissolved in 0.9% (w/v) physiological saline to give a bacterial turbidimeter with an indication of 0.50 Maillard turbidity units at a concentration of 1X 10 8 cfu/mL. 100. Mu.L of the above bacterial suspension was pipetted into a culture medium and rapidly spread on a petri dish. Then, the nano-particle membrane carrying 5 mu L is tightly attached to the surface of a culture medium by using sterile forceps, and after culturing for 24 hours at 37 ℃, the diameter of a bacteriostasis ring is measured to be 14.7mm, and the test result is shown in Table 3.
TABLE 3 Table 3
The range of the inhibition zone of the beta-acid to staphylococcus aureus is 7.20-14.93mm; the range of the bacteriostasis zone for the escherichia coli is as follows: 7.27-12.37mm. In contrast, the ranges of the beta-acid loaded chitosan nanoparticle on staphylococcus aureus and escherichia coli inhibition zones are respectively as follows: 7.23-14.70mm and 7.17-11.57mm; under the comprehensive 4-6 experiment, the drug effect of the beta-acid loaded chitosan nano-particles is better for the same bacteria under the condition of the same drug loading rate.
8. Anticancer agent
Colorectal cancer cells (HCT-116 and HT-29) were taken and seeded in 96-well plates (1X 10 per well) 4 Density of cells). The cells were allowed to grow for 6 hours for cell attachment. The culture medium in the 96-well plate is replaced by the drug-containing culture medium (80, 40, 20, 10, 5 mug/mL) for 24h, and the times are thatThe drug-containing medium was aspirated daily, fresh medium was changed, 10. Mu.L of CCK-8 solution was added to each well, incubated at 37℃for 1h, and the Optical Density (OD) was measured per well using enzyme labeling at 450 nm. The test results are shown in table 4.
TABLE 4 Table 4
IC of beta-acid to HCT-116 50 2.75 μg/mL; IC for HT-29 50 10.22. Mu.g/mL. In contrast, β -acid loaded chitosan nanoparticle IC to HCT-116 and HT-29 50 The ranges are respectively as follows: 8.12-40.12 mug/mL and 14.92-959.9 mug/mL, the anticancer effect of the chitosan nanoparticle loaded with beta-acid is better.
The foregoing disclosure is illustrative of the preferred embodiments of the present invention, and is not to be construed as limiting the scope of the invention, as it is understood by those skilled in the art that all or part of the above-described embodiments may be practiced with equivalents thereof, which fall within the scope of the invention as defined by the appended claims.
Claims (10)
1. A preparation method of a beta-acid loaded chitosan nanomaterial is characterized by comprising the following steps of: comprises the following steps of the method,
s1: dissolving chitosan in acetic acid solution to obtain chitosan solution, and dissolving beta-acid in absolute ethyl alcohol to obtain beta-acid solution;
s2: mixing the beta-acid solution with the chitosan solution to obtain a mixed solution;
s3: dropwise adding a sodium tripolyphosphate solution into the mixed solution to perform ionic gel method reaction for a preset time to generate a chitosan nanoparticle solution containing beta-acid;
s4: and centrifuging and freeze-drying the chitosan nanoparticle solution containing the loaded beta-acid to obtain the chitosan nanoparticle loaded with the beta-acid.
2. The method for preparing the beta-acid loaded chitosan nanomaterial of claim 1, wherein the method comprises the following steps: in the step S1, the concentration of the chitosan solution is 1mg/mL-10mg/mL, and the concentration of the beta-acid solution is 1mg/mL-30mg/mL.
3. The method for preparing the beta-acid loaded chitosan nanomaterial of claim 2, wherein the method comprises the following steps: in the step S2, the volume ratio of the beta-acid solution to the chitosan solution is 1:0.25-1.5.
4. The method for preparing the beta-acid loaded chitosan nanomaterial of claim 1, wherein the method comprises the following steps: in the step S2, when the beta-acid solution and the chitosan solution are mixed, the mixed solution is obtained by stirring for 10-30 min under the normal temperature condition.
5. The method for preparing the beta-acid loaded chitosan nanomaterial of claim 1, wherein the method comprises the following steps: in the step S3, the concentration of the sodium tripolyphosphate solution is 1mg/mL-10mg/mL.
6. The method for preparing the beta-acid loaded chitosan nanomaterial according to claim 4, wherein the method comprises the following steps: in the step S3, the mass ratio of the sodium tripolyphosphate solution to the mixed solution is 1:0.5-2.
7. The method for preparing the beta-acid loaded chitosan nanomaterial of claim 1, wherein the method comprises the following steps: in the step S3, dropwise adding a sodium tripolyphosphate solution into the mixed solution at normal temperature, and reacting for a preset time of 1-2h.
8. A beta-acid-loaded chitosan nanomaterial prepared by the method for preparing a beta-acid-loaded chitosan nanomaterial of any one of claims 1 to 7.
9. The use of the beta-acid loaded chitosan nanomaterial of claim 8 in the preparation of antibacterial or anticancer drugs.
10. The use of the beta-acid loaded chitosan nanomaterial of claim 9 in the preparation of a medicament for inhibiting staphylococcus aureus or escherichia coli or anti-colon cancer.
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