CN104013682B - A kind of Traditional Chinese medicine bacteriostatic composition suppressing micrococcus luteus - Google Patents

Abstract

The invention discloses a kind of Traditional Chinese medicine bacteriostatic composition suppressing micrococcus luteus, belong to the field of Chinese medicines.In bacteria inhibiting composition, Radix Et Rhizoma Rhei extract mixes with ratio with water-soluble nano silver, and share of two kinds of medicines plays collaborative effect to suppression micrococcus luteus.A kind of Radix Et Rhizoma Rhei extract of the present invention and nano-silver bacteriostatic composition are compared with prior art, solve the problem of Radix Et Rhizoma Rhei antibacterial large usage quantity in conventional bacteriostasis method, adopt simultaneously the antibacterial substance of two kinds of low dosages decrease microorganism drug resistance Probability, reduce its toxicity to human body, more safe.

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 comprises the following steps: adding water in an amount 9 times the weight of the rhubarb, extracting in a water bath at 95°C for 4 hours, and then concentrating under reduced pressure to obtain the extract. The resulting 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 particle is 3-10nm, and the surface of the nano-silver is coated with an amphoteric polymer.

The water-soluble nano-silver has a particle size of 3-10nm and a carboxyl group on the outside. It is prepared according to the following method: dissolve 22.8g of myristic acid in 140mL of methanol and water mixed in a ratio of 2:5, and add 4g of sodium hydroxide to the solution Precipitate, filter, and add the precipitate to 100mL concentration of 10mol/L water-soluble silver nitrate solution to obtain silver myristate; take 6.7g concentration of 20mmoL/L silver myristate in a 100mL beaker, and add to the beaker Add 58mL of triethylamine with a concentration of 40mmoL/L to the solution, stir electromagnetically at 80°C for 2h, the white silver myristate powder gradually turns brown, and the insoluble precursor disappears, add 20mL of acetone to precipitate, filter with acetone After washing the precipitate several times, it is 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 The inhibitory effect of 200 μL rhubarb extract on Escherichia coli O157:H7, Figure 1b The inhibitory effect of 2 μL nano-silver on Escherichia coli O157:H7, Figure 1c The synergistic antibacterial effect of 100 μL rhubarb extract and 1 μL nano-silver on Escherichia coli O157: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 The inhibitory effect of 150 μL rhubarb extract on Escherichia coli O157:H7, Figure 2b The inhibitory effect of 2 μL nano-silver on Escherichia coli O157:H7, Figure 2c The synergistic antibacterial effect of 75 μL rhubarb extract and 1 μL nano-silver on Escherichia coli O157: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 M. luteus, Figure 3b The inhibitory effect of 16 μL nano-silver on M. luteus, Figure 3c The synergistic antibacterial effect of 400 μL rhubarb extract and 8 μL nano-silver on M. luteus.

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. luteus, Figure 4c The synergistic antibacterial effect of 200 μL rhubarb extract and 8 μL nano-silver on M. luteus.

Detailed ways

Example 1

1. The preparation process of rhubarb extract:

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

2. Preparation of water-soluble silver nanoparticles

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

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 and place it in 10 mL of LB broth medium, culture it in a 37°C incubator for 16 hours, and then inoculate it into 5 mL of LB broth medium at an inoculum size of 1%. After incubation at 37°C for 4 h. Take 1mL 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, add 200 μL of rhubarb extract (emodin content is 1.92 μg/g, the same below) to No. 1 centrifuge tube, and centrifuge to No. 2 Add 2 μL of water-soluble nano-silver with a concentration of 10 mg/mL to the tube, add 100 μL of rhubarb extract and 1 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 without adding any of the above antibacterial agent. In order to ensure that the final volume of each tube of liquid is equal, the difference is made up with 0.1% peptone water, and placed in a shaker incubator at 37°C for 2 hours. 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 Escherichia coli O157:H7, Figure 1b shows the inhibitory effect of 2 μL rhubarb extract on Escherichia coli O157:H7, Figure 1c shows the inhibitory effect of 100 μL rhubarb extract and 1 μL nano silver on Escherichia coli O157: Synergistic antibacterial effect of 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 and place it in 10 mL of LB broth medium, culture it in a 37°C incubator for 16 hours, and then inoculate it into 5 mL of LB broth medium at an inoculum size of 1%. After incubation at 37°C for 4 h. Take 1mL 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 water-soluble nano For silver, add 75 μL of rhubarb extract and 1 μL of water-soluble nano-silver with a concentration of 10 mg/mL to No. 3 centrifuge tube, and No. 4 centrifuge tube is used as a blank control without adding any of the above-mentioned antibacterial agents. In order to ensure that the final volume of each tube of liquid is equal, the difference is made up with 0.1% peptone water, and placed in a shaker incubator at 37°C for 2 hours. 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 Escherichia coli O157:H7, Figure 2b shows the inhibitory effect of 2 μL rhubarb extract on Escherichia coli O157:H7, and Figure 2c shows the inhibitory effect of 75 μL rhubarb extract and 1 μL nanosilver on Escherichia coli O157: Synergistic antibacterial effect of 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, put it in 5mL beef extract peptone liquid medium, culture it in a 30°C incubator for 24 hours, and then inoculate 5mL of beef extract peptone according to the inoculation amount of 1%. In liquid medium, cultured at 30°C for 12h. Take 1mL 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 1mL of the above-mentioned diluted bacterial solution into 1.5mL sterilized centrifuge tubes, add 800μL of rhubarb extract (500μL of bacteria solution only) to No. 400 μL of rhubarb extract and 8 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-mentioned 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 hours, and 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 from three gradients of 10 ^-2 , 10 ^-3 , and 10 ^-4 respectively. After drying the plate, culture it upside down at 30°C for 48 hours, 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 Micrococcus luteus, Figure 3b shows the inhibitory effect of 16 μL nano-silver on Micrococcus luteus, and Figure 3c shows the synergistic antibacterial effect of 400 μL rhubarb extract and 8 μL nano-silver on Micrococcus luteus effect.

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 beef extract peptone agar medium, put it in 5mL beef extract peptone liquid medium, culture it in a 30°C incubator for 24 hours, and then inoculate 5mL of beef extract peptone according to the inoculation amount of 1%. In liquid medium, cultured at 30°C for 12h. Take 1mL 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 water-soluble nano Silver, add 200 μL of rhubarb extract and 8 μL of water-soluble nano-silver to No. 3 centrifuge tube, and No. 4 centrifuge tube is used as a blank control without adding any of the above-mentioned 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 hours, and 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 from three gradients of 10 ^-2 , 10 ^-3 , and 10 ^-4 respectively. After drying the plate, culture it upside down at 30°C for 48 hours, 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 Micrococcus luteus, Figure 4b shows the inhibitory effect of 16 μL nano-silver on Micrococcus luteus, and Figure 4c shows the synergistic antibacterial effect of 200 μL rhubarb extract and 8 μL nano-silver on Micrococcus luteus effect.

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. suppress a Traditional Chinese medicine bacteriostatic composition for micrococcus luteus, it is characterized in that being made up 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 4h, then obtain extracting solution through concentrating under reduced pressure through 95 DEG C of water-baths; Described water-soluble nano silver is prepared according to following method: get 22.8g tetradecanoic acid and be dissolved in the first alcohol and water that 140mL mixes in 2:5 ratio, the sodium hydroxide adding 4g in solution separates out precipitation, filter, precipitation being joined 100mL concentration is obtain tetradecanoic acid silver in the water miscible silver nitrate solution of 10mol/L; Taking 6.7g concentration is that 20mmoL/L tetradecanoic acid silver is in 100mL beaker, the triethylamine that 58mL concentration is 40mmoL/L is added in beaker, electromagnetic agitation 2h at 80 DEG C, the tetradecanoic acid silver powder of white becomes brown, insoluble disappearance gradually, adds 20mL acetone precipitation and separates out, sucking filtration, after washing with acetone precipitation several, vacuum drying, namely obtains nano silver particles powder; The volume ratio of Radix Et Rhizoma Rhei extract, water-soluble nano silver is 25 ~ 50:1.

2. Traditional Chinese medicine bacteriostatic composition as claimed in claim 1, is characterized in that gained extracting solution is through 0.22 μm of membrane filtration.

3. Traditional Chinese medicine bacteriostatic composition as described in as arbitrary in claim 1 ~ 2 suppresses the application in micrococcus luteus medicine in preparation.