CN104013682A - Traditional Chinese medicine antibacteria composition for inhibiting micrococcus luteus - Google Patents

Abstract

The invention discloses a traditional Chinese medicine antibacteria composition for inhibiting micrococcus luteus, and belongs to the traditional Chinese medicine field. A rheum officinale extracting solution and water-soluble nano-silver are mixed in the antibacteria composition in proportion, two medicines are synthesized to play the synergistic effect to inhibit the micrococcus luteus. Compared with the prior art, the antibacteria composition containing the rheum officinale extract and the nano-silver can solve the problem that the dosage of the rheum officinale bacteriostatic agent in the conventional antibacteria method is great; and meanwhile, the microorganism medicine resistance generating rate is reduced by adopting two antibacterial substances in low dosage, the toxicity to the human body is lowered, and the composition is safer.

Description

A traditional Chinese medicine antibacterial composition for inhibiting Micrococcus luteus

technical field

The invention belongs to the field of traditional Chinese medicines, in particular to a traditional Chinese medicine antibacterial composition.

Background technique

Rhubarb is a collective name for various perennial plants of the Polygonaceae Rhubarb genus, and it is also the name of Chinese medicinal materials. Because it likes a cool climate and is resistant to cold, it mainly grows in the damp environment on the edge of mountain forests or grass slopes. The traditional Chinese medicine rhubarb has the functions of attacking stagnation, clearing damp and heat, purging fire, cooling blood, removing blood stasis, and detoxifying. Modern pharmacological studies have proved that rhubarb has antibacterial effects, including Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, Salmonella typhi, Shigella, hemolytic streptococcus, pneumococcus, diphtheria bacillus, Bacillus anthracis, Proteus, etc. Strong inhibitory effect, in addition, it also has the effects of anti-tumor, lowering blood pressure, invigorating the stomach, promoting gallbladder, protecting the liver, strengthening the heart, delaying aging, and regulating immune function. Rhubarb has attracted people's attention because of its high medicinal and health value.

Concentrated Chinese medicine preparations made into oral liquids or antibacterial agents, coating agents, etc. have a large dosage at one time, and long-term use will affect human health.

Contents of the invention

Aiming at the large amount of rhubarb bacteriostatic agent once used and the long-term use affecting human health, the inventor found through a large number of experiments that nano-silver can effectively enhance the antibacterial performance of rhubarb extract, reduce the amount of rhubarb bacteriostatic agent, and use two low-dose The antibacterial composition is safer. The object of the present invention is to provide a rhubarb nano-silver bacteriostatic composition, which can reduce the once-used dose of rhubarb bacteriostatic agent and reduce the toxicity of medicine to human body.

The present invention provides following technical scheme:

An antibacterial composition, containing rhubarb extract and water-soluble nano-silver;

The preparation of the rhubarb extract includes the following steps: adding 9 times the weight of rhubarb, extracting in a water bath at 95 °C for 4 h, and then concentrating under reduced pressure to obtain the extract. The obtained extract was filtered through a 0.22 μm membrane filter. Finally, the extract was subjected to standard quantification of emodin by high performance liquid chromatography.

The particle size of the water-soluble nano-silver particles is 3-10 nm, and the surface of the nano-silver is coated with an amphoteric polymer.

The particle size of water-soluble nano-silver is 3-10 nm, and the appearance is carboxyl group. It is prepared according to the following method: take 22.8 g of myristic acid and dissolve it in 140 mL of methanol and water mixed in a ratio of 2:5, and add 4 g of Sodium hydroxide precipitates, filters, and the precipitate is added to 100 mL of 10 mol/L water-soluble silver nitrate solution to obtain silver myristate; 6.7 g of 20 mmoL/L silver myristate is weighed in In a 100 mL beaker, add 58 mL of triethylamine with a concentration of 40 mmoL/L to the beaker, and stir it electromagnetically for 2 h at 80 °C. The white silver myristate powder gradually turns brown, and the insoluble precursor disappears. Add 20 mL of acetone was precipitated, filtered by suction, washed with acetone several times, and dried in vacuum to obtain nano-silver particle powder. The water-soluble nano-silver particle prepared according to the method has good synergistic antibacterial effect with rhubarb extract.

When inhibiting Escherichia coli O157:H7, the volume ratio of rhubarb extract and water-soluble nano-silver is 75-100:1

When inhibiting Micrococcus luteus, the volume ratio of rhubarb extract and water-soluble nano-silver is 25-50:1.

The present invention also relates to the application of the above antibacterial composition in inhibiting Escherichia coli O157:H7.

The present invention also relates to the application of the above antibacterial composition in inhibiting Micrococcus luteus

The beneficial effects of the present invention are: the application of water-soluble nano-silver can effectively enhance the rhubarb bacteriostatic performance, reduce the consumption of rhubarb bacteriostatic agents, and what is unexpected is that the combined use of two kinds of medicines can inhibit Escherichia coli O157:H7 and Micrococcus luteus plays a synergistic effect. At the same time, the antibacterial composition uses two low-dose antibacterial substances, which is safer than the use of high-dose rhubarb or water-soluble nano-silver, and can be used as a safe and reliable external coating agent etc.

Description of drawings

Figure 1 The ratio of rhubarb extract to nano-silver is 100:1 for synergistic antibacterial effect on Escherichia coli O157:H7

Figure 1a Inhibitory effect of 200 μL rhubarb extract on E. coli O157:H7, Figure 1b Inhibition of 2 μL nano-silver on E. coli O157:H7, Figure 1c 100 μL rhubarb extract and 1 μL nano-silver on E. coli O157 : Synergistic antibacterial effect of H7.

Figure 2 The ratio of rhubarb extract to nano-silver is 75:1 for synergistic antibacterial effect on Escherichia coli O157:H7

Figure 2a Inhibitory effect of 150 μL rhubarb extract on E. coli O157:H7, Figure 2b Inhibition of 2 μL nano-silver on E. coli O157:H7, Figure 2c 75 μL rhubarb extract and 1 μL nano-silver on E. coli O157 : Synergistic antibacterial effect of H7.

Figure 3 The ratio of rhubarb extract to nano-silver is 50:1 for synergistic antibacterial effect on Micrococcus luteus

Figure 3a The inhibitory effect of 800 μL rhubarb extract on Micrococcus luteus, Figure 3b The inhibitory effect of 16 μL nano-silver on M. Bacteria.

Figure 4 The ratio of rhubarb extract to nano-silver is 25:1 for synergistic antibacterial effect on Micrococcus luteus

Figure 4a The inhibitory effect of 400 μL rhubarb extract on M. luteus, Figure 4b The inhibitory effect of 16 μL nano-silver on M. Bacteria.

Detailed ways

Example 1

1. The preparation process of rhubarb extract:

Weigh 50 g of rhubarb, add 450 mL of water, extract at 95 °C for 4 h, pass through a vacuum rotary concentrator, use a 0.22 μm filter to remove impurities and bacteria in the extract, and finally use high performance liquid chromatography to measure The emodin content in the extract was 1.92 μg/g.

2. Preparation of water-soluble silver nanoparticles

Take 22.8 g of myristic acid and dissolve it in 140 mL of methanol and water mixed at a ratio of 2:5, add 4 g of sodium hydroxide to the solution to precipitate a precipitate, filter it, and add the precipitate to 100 mL of water-soluble solution with a concentration of 10 mol/L. Silver myristate was obtained from a neutral silver nitrate solution; 6.7 g of silver myristate with a concentration of 20 mmoL/L was weighed in a 100 mL beaker, and 58 mL of triethylamine with a concentration of 40 mmoL/L was added to the beaker , electromagnetically stirred at 80°C for 2 h, the white silver myristate powder gradually turned brown, and the insoluble precursor disappeared, and 20 mL of acetone was added to precipitate out, filtered by suction, washed with acetone for several times, and dried in vacuum. The nano silver particle powder is obtained.

3. The synergistic antibacterial effect of rhubarb extract and water-soluble nano-silver.

a. Culture of bacteria

Pick a single colony of Escherichia coli O157:H7 on LB agar medium, place it in 10 mL LB broth medium, culture it in a 37 °C incubator for 16 h, and then inoculate it into 5 mL LB at an inoculum size of 1%. After incubation in broth for 4 h at 37 °C. Take 1 mL of the above bacterial solution and serially dilute three gradients with 0.1% peptone water, and the final bacterial solution is about 10 ⁶ CFU/mL.

b. The synergistic effect of water-soluble nano-silver and rhubarb extract on antibacterial properties

Take 1 mL of the above-mentioned diluted bacterial solution into 1.5 mL sterilized centrifuge tubes, respectively add 200 μL of rhubarb extract (emodin content is 1.92 μg/g, the same below) to No. 1 centrifuge tube, and add to 2 Add 2 μL of water-soluble nano-silver with a concentration of 10 mg/mL to No. 3 centrifuge tube, add 100 μL rhubarb extract and 1 μL of 10 mg/mL water-soluble nano-silver to No. Blank control, without adding any of the above antibacterial agents. In order to ensure that the final volume of each tube of liquid was equal, the difference was made up with 0.1% peptone water, and placed in a shaker incubator at 37°C for 2 h. Each experiment was performed in parallel three times.

c, plate count

The solution to be tested in the experimental group was serially diluted in two gradients with PBS, and 200 μL of each of 10 ⁰ , 10 ^-1 , and 10 ^-2 was taken out and spread evenly on the

On the LB solid plate; the blank group was serially diluted with four gradients, and the three gradients from 10 ^-2 , 10 ^-3 , and 10 ^-4 were all coated on LB

on a solid plate. After drying the plate, culture it upside down at 37°C for 12 hours, count the colonies after they grow out, and form a single colony with 30-300 colonies.

Bits (CFU) are valid counting ranges. The number of viable bacteria per milliliter of the original bacterial solution = plate count * dilution factor * 5

The experimental results are as follows:

antibacterial substances The number of colonies that can be counted after adding the antibacterial agent blank 6.5×10 ⁷ CFU/mL 200 μL of rhubarb extract 2.6×10 ⁶ CFU/mL Contains 2 μL of silver nanoparticles at a concentration of 10 mg/mL 4.8×10 ² CFU/mL Contains 100 μL of rhubarb extract and 1 μL of silver nanoparticles at a concentration of 10 mg/mL 0 CFU/mL

As shown in Figure 1, Figure 1a shows the inhibitory effect of 200 μL rhubarb extract on E. coli O157:H7, Figure 1b shows the inhibitory effect of 2 μL of nano-silver on E. Synergistic antibacterial effect of silver nanoparticles against Escherichia coli O157:H7.

Experiments show that the bacteriostatic composition provided by the invention has the effect of reducing the one-time dosage of rhubarb bacteriostatic agents, and the combination of the two drugs has a synergistic effect on inhibiting Escherichia coli O157:H7.

Example 2

1. The preparation process of rhubarb extract:

With embodiment 1.

2. Preparation of water-soluble silver nanoparticles

With embodiment 1.

3. The synergistic antibacterial effect of rhubarb extract and water-soluble nano-silver.

a. Culture of bacteria

Pick a single colony of Escherichia coli O157:H7 on LB agar medium, place it in 10 mL LB broth medium, culture it in a 37 °C incubator for 16 h, and then inoculate it into 5 mL LB at an inoculum size of 1%. After incubation in broth for 4 h at 37 °C. Take 1 mL of the above bacterial solution and serially dilute three gradients with 0.1% peptone water, and the final bacterial solution is about 10 ⁶ CFU/mL.

b. The synergistic effect of water-soluble nano-silver and rhubarb extract on antibacterial properties

Take 1 mL of the above-mentioned diluted bacterial solution in 1.5 mL sterilized centrifuge tubes, add 150 μL of rhubarb extract to No. 1 centrifuge tube, and add 2 μL of rhubarb extract to No. 75 μL of rhubarb extract and 1 μL of water-soluble nano-silver with a concentration of 10 mg/mL were added to No. 3 centrifuge tube, and No. 4 centrifuge tube was used as a blank control without adding any of the above antibacterial agents. In order to ensure that the final volume of each tube of liquid was equal, the difference was made up with 0.1% peptone water, and placed in a shaker incubator at 37°C for 2 h. Each experiment was performed in parallel three times.

c, plate count

The solution to be tested in the experimental group was serially diluted in two gradients with PBS, and 200 μL of each of 10 ⁰ , 10 ^-1 , and 10 ^-2 was taken out and spread evenly on the

On the LB solid plate; the blank group was serially diluted with four gradients, and the three gradients from 10 ^-2 , 10 ^-3 , and 10 ^-4 were all coated on LB

on a solid plate. After drying the plate, culture it upside down at 37°C for 12 hours, count the colonies after they grow out, and form a single colony with 30-300 colonies.

Bits (CFU) are valid counting ranges. The number of viable bacteria per milliliter of the original bacterial solution = plate count * dilution factor * 5

The experimental results are as follows:

antibacterial substances The number of colonies that can be counted after adding the antibacterial agent blank 5.7×10 ⁶ CFU/mL 150 μL of rhubarb extract 2.6×10 ⁶ CFU/mL Contains 2 μL of silver nanoparticles at a concentration of 10 mg/mL 4.8×10 ² CFU/mL Contains 75 μL rhubarb extract, 1 μL nano-silver solution with a concentration of 10 mg/mL 200 CFU/mL

As shown in Figure 2, Figure 2a shows the inhibitory effect of 150 μL rhubarb extract on E. coli O157:H7, Figure 2b shows the inhibitory effect of 2 μL of nano-silver on E. Synergistic antibacterial effect of silver nanoparticles against Escherichia coli O157:H7.

Experiments show that the bacteriostatic composition provided by the invention has the effect of reducing the one-time dosage of rhubarb bacteriostatic agents, and the combination of the two drugs has a synergistic effect on inhibiting Escherichia coli O157:H7.

Example 3

1. The preparation process of rhubarb extract:

With embodiment 1.

2. Preparation of water-soluble silver nanoparticles

With embodiment 1.

3. The synergistic antibacterial effect of rhubarb extract and water-soluble nano-silver.

a. Culture of bacteria

Pick a single colony of Micrococcus luteus on the beef extract-peptone agar medium and put it in 5 mL of beef extract-peptone liquid medium, culture it in a 30 °C incubator for 24 h, and then inoculate it in 5 mL of After cultured in beef extract peptone liquid medium at 30 °C for 12 h. Take 1 mL of the above bacterial solution and serially dilute three gradients in the mixture of PBS and liquid medium at a ratio of 4:1, and the final bacterial solution is about 10 ⁶ -10 ⁷ CFU/mL.

b. The synergistic effect of water-soluble nano-silver and rhubarb extract on antibacterial properties

Take 1 mL of the above-mentioned diluted bacterial solution into 1.5 mL sterilized centrifuge tubes, add 800 μL of rhubarb extract (500 μL of bacteria solution only) to No. 1 centrifuge tube, and add Add 16 μL of water-soluble nano-silver with a concentration of 10 mg/mL, add 400 μL of rhubarb extract and 8 μL of water-soluble nano-silver with a concentration of 10 mg/mL to No. 3 centrifuge tube, and No. 4 centrifuge tube as a blank control. Do not add any of the above bacteriostatic agents. In order to ensure that the final volume of each tube of liquid is equal, the difference is made up with a mixture of PBS and liquid medium at a ratio of 4:1. Placed in a shaker incubator at 30°C for 6 h, each experiment was repeated three times.

c, plate count

The solution to be tested in the experimental group was serially diluted in two gradients with PBS, and 100 μL of each of 10 ^-1 , 10 ^-2 , and 10 ^-3 was taken out and spread evenly.

Spread on the beef extract peptone solid plate; the blank group was serially diluted with four gradients, and spread evenly on the beef extract peptone solid plate respectively from three gradients of 10 ^-2 , 10 ^-3 , and 10 ^-4 . After drying the plate, culture it upside down at 30°C for 48 h, count the colonies after they grow out, and take 20-200 colony forming units (CFU) as the effective counting range. The number of viable bacteria per milliliter of original bacterial solution = plate count * dilution factor * 10

antibacterial substances The number of colonies that can be counted after adding the antibacterial agent blank 1.4×10 ⁷ CFU/mL 800 μL of rhubarb extract 2.1×10 ⁶ CFU/mL Contains 16 μL of silver nanoparticles at a concentration of 10 mg/mL 1.3×10 ⁶ CFU/mL Including 400 μL of rhubarb extract and 8 μL of silver nanoparticles at a concentration of 10 mg/mL 3.5×10 ⁵ CFU/mL

As shown in Figure 3, Figure 3a shows the inhibitory effect of 800 μL rhubarb extract on M. Synergistic antibacterial effect of Micrococcus luteus.

Experiments show that the bacteriostatic composition provided by the invention has the effect of reducing the dosage of the rhubarb bacteriostatic agent at one time, and the combined use of the two drugs has a synergistic effect on inhibiting Micrococcus luteus.

Example 4

1. The preparation process of rhubarb extract:

With embodiment 1.

2. Preparation of water-soluble silver nanoparticles

With embodiment 1.

4. Synergistic antibacterial effect of rhubarb extract and water-soluble nano-silver.

a. Culture of bacteria

Pick a single colony of Micrococcus luteus on the peptone agar medium of beef extract and put it in 5 mL of peptone liquid medium of beef extract, culture it in a 30 °C incubator for 24 h, and then inoculate 5 mL of beef at an inoculation amount of 1%. Incubate peptone broth for 12 h at 30 °C. Take 1 mL of the above bacterial solution and serially dilute three gradients in the mixture of PBS and liquid medium at a ratio of 4:1, and the final bacterial solution is about 10 ⁶ -10 ⁷ CFU/mL.

b. The synergistic effect of water-soluble nano-silver and rhubarb extract on antibacterial properties

Take 1 mL of the above-mentioned diluted bacterial solution in 1.5 mL sterilized centrifuge tubes, add 400 μL of rhubarb extract to No. 1 centrifuge tube, and add 16 μL of rhubarb extract to No. 200 μL of rhubarb extract and 8 μL of water-soluble nano-silver were added to No. 3 centrifuge tube, and No. 4 centrifuge tube was used as a blank control without adding any of the above antibacterial agents. In order to ensure that the final volume of each tube of liquid is equal, the difference is made up with a mixture of PBS and liquid medium at a ratio of 4:1. Placed in a shaker incubator at 30°C for 6 h, each experiment was repeated three times.

c. Plate count

The solution to be tested in the experimental group was serially diluted in two gradients with PBS, and 100 μL of each of 10 ^-1 , 10 ^-2 , and 10 ^-3 was taken out and spread evenly.

Spread on the beef extract peptone solid plate; the blank group was serially diluted with four gradients, and spread evenly on the beef extract peptone solid plate respectively from three gradients of 10 ^-2 , 10 ^-3 , and 10 ^-4 . After drying the plate, culture it upside down at 30°C for 48 h, count the colonies after they grow out, and take 20-200 colony forming units (CFU) as the effective counting range. The number of viable bacteria per milliliter of original bacterial solution = plate count * dilution factor * 10

antibacterial substances The number of colonies that can be counted after adding the antibacterial agent blank 1.6×10 ⁷ CFU/mL 400 μL of rhubarb extract 2.6×10 ⁶ CFU/mL Contains 16 μL of silver nanoparticles at a concentration of 10 mg/mL 1.54×10 ⁶ CFU/mL Including 200 μL of rhubarb extract and 8 μL of silver nanoparticles at a concentration of 10 mg/mL 6.5×10 ⁵ CFU/mL

As shown in Figure 4, Figure 4a shows the inhibitory effect of 400 μL rhubarb extract on M. luteus, Figure 4b shows the inhibitory effect of 16 μL of nano-silver on M. Synergistic antibacterial effect of Micrococcus luteus.

Experiments show that the bacteriostatic composition provided by the invention has the effect of reducing the dosage of the rhubarb bacteriostatic agent at one time, and the combined use of the two drugs has a synergistic effect on inhibiting Micrococcus luteus.

Claims (3)

1. a Chinese medicine bacteria inhibiting composition that suppresses micrococcus luteus, is characterized in that being comprised of Radix Et Rhizoma Rhei extract, water-soluble nano silver; Described Radix Et Rhizoma Rhei extract preparation comprises the steps: that amount of water is 9 times of Radix Et Rhizoma Rhei weight, extracts 4 h, then obtain extracting solution through concentrating under reduced pressure through 95 ℃ of water-baths; Described water-soluble nano silver is prepared according to following method: get 22.8 g tetradecanoic acids and be dissolved in 140 mL in the first alcohol and water of 2:5 ratio mixing, in solution, add the sodium hydroxide of 4 g to separate out precipitation, filter, it is to obtain tetradecanoic acid silver in the water miscible silver nitrate solution of 10 mol/L that precipitation is joined to 100 mL concentration; Taking 6.7 g concentration is that 20 mmoL/L tetradecanoic acid silver are in 100 mL beakers, to adding 58 mL concentration in beaker, it is the triethylamine of 40 mmoL/L, electromagnetic agitation 2 h at 80 ℃, the tetradecanoic acid silver powder of white becomes brown, insoluble disappearance gradually, adds 20 mL acetone precipitations to separate out, sucking filtration, with after washing with acetone precipitation several, vacuum drying, obtains nano silver particles powder; The volume ratio of Radix Et Rhizoma Rhei extract, water-soluble nano silver is and 25～50:1.

2. Chinese medicine bacteria inhibiting composition as claimed in claim 1, is characterized in that gained extracting solution is through 0.22 μ m membrane filtration.

3. as described in as arbitrary in claim 1 ~ 2, Chinese medicine bacteria inhibiting composition suppresses the application in micrococcus luteus medicine in preparation.