CN103977095B - A kind of Rhizoma Coptidis extract and nano-silver bacteriostatic composition suppressing micrococcus luteus - Google Patents
A kind of Rhizoma Coptidis extract and nano-silver bacteriostatic composition suppressing micrococcus luteus Download PDFInfo
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- 239000000284 extract Substances 0.000 title claims abstract description 66
- BQCADISMDOOEFD-UHFFFAOYSA-N Silver Chemical compound [Ag] BQCADISMDOOEFD-UHFFFAOYSA-N 0.000 title claims abstract description 63
- 241000191938 Micrococcus luteus Species 0.000 title claims abstract description 25
- 239000000203 mixture Substances 0.000 title claims abstract description 22
- 230000003385 bacteriostatic effect Effects 0.000 title claims abstract description 7
- 241000894006 Bacteria Species 0.000 claims abstract description 39
- 230000002401 inhibitory effect Effects 0.000 claims abstract description 13
- 239000003814 drug Substances 0.000 claims abstract description 11
- 238000000034 method Methods 0.000 claims abstract description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 16
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 claims description 12
- YBHILYKTIRIUTE-UHFFFAOYSA-N berberine Chemical compound C1=C2CC[N+]3=CC4=C(OC)C(OC)=CC=C4C=C3C2=CC2=C1OCO2 YBHILYKTIRIUTE-UHFFFAOYSA-N 0.000 claims description 12
- 229940093265 berberine Drugs 0.000 claims description 12
- QISXPYZVZJBNDM-UHFFFAOYSA-N berberine Natural products COc1ccc2C=C3N(Cc2c1OC)C=Cc4cc5OCOc5cc34 QISXPYZVZJBNDM-UHFFFAOYSA-N 0.000 claims description 12
- 238000001556 precipitation Methods 0.000 claims description 12
- 238000002360 preparation method Methods 0.000 claims description 12
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 9
- ZMANZCXQSJIPKH-UHFFFAOYSA-N Triethylamine Chemical compound CCN(CC)CC ZMANZCXQSJIPKH-UHFFFAOYSA-N 0.000 claims description 9
- FDBMFXMFZNNOQV-UHFFFAOYSA-N silver;tetradecanoic acid Chemical compound [Ag].CCCCCCCCCCCCCC(O)=O FDBMFXMFZNNOQV-UHFFFAOYSA-N 0.000 claims description 9
- 238000005374 membrane filtration Methods 0.000 claims description 6
- 239000000843 powder Substances 0.000 claims description 6
- SQGYOTSLMSWVJD-UHFFFAOYSA-N silver(1+) nitrate Chemical compound [Ag+].[O-]N(=O)=O SQGYOTSLMSWVJD-UHFFFAOYSA-N 0.000 claims description 6
- TUNFSRHWOTWDNC-UHFFFAOYSA-N tetradecanoic acid Chemical compound CCCCCCCCCCCCCC(O)=O TUNFSRHWOTWDNC-UHFFFAOYSA-N 0.000 claims description 6
- 239000002245 particle Substances 0.000 claims description 4
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 3
- 238000013019 agitation Methods 0.000 claims description 3
- 230000008034 disappearance Effects 0.000 claims description 3
- 238000001914 filtration Methods 0.000 claims description 3
- 239000012567 medical material Substances 0.000 claims description 3
- 229910001961 silver nitrate Inorganic materials 0.000 claims description 3
- 238000001291 vacuum drying Methods 0.000 claims description 3
- 238000005406 washing Methods 0.000 claims description 3
- 230000000844 anti-bacterial effect Effects 0.000 abstract description 49
- 230000001629 suppression Effects 0.000 abstract description 13
- 230000000694 effects Effects 0.000 abstract description 12
- 229940126678 chinese medicines Drugs 0.000 abstract description 2
- 239000000126 substance Substances 0.000 abstract description 2
- 230000001988 toxicity Effects 0.000 abstract description 2
- 231100000419 toxicity Toxicity 0.000 abstract description 2
- 206010059866 Drug resistance Diseases 0.000 abstract 1
- 244000005700 microbiome Species 0.000 abstract 1
- 239000007788 liquid Substances 0.000 description 25
- 229910052709 silver Inorganic materials 0.000 description 20
- 239000004332 silver Substances 0.000 description 20
- 239000000243 solution Substances 0.000 description 17
- 241001333951 Escherichia coli O157 Species 0.000 description 16
- 239000001888 Peptone Substances 0.000 description 14
- 238000013207 serial dilution Methods 0.000 description 12
- 235000015278 beef Nutrition 0.000 description 10
- 238000002474 experimental method Methods 0.000 description 9
- 239000012530 fluid Substances 0.000 description 8
- 239000002609 medium Substances 0.000 description 8
- 239000007787 solid Substances 0.000 description 8
- 239000003708 ampul Substances 0.000 description 5
- 229940079593 drug Drugs 0.000 description 5
- 239000001963 growth medium Substances 0.000 description 5
- 238000004128 high performance liquid chromatography Methods 0.000 description 5
- 108010080698 Peptones Proteins 0.000 description 4
- 230000001332 colony forming effect Effects 0.000 description 4
- 239000004744 fabric Substances 0.000 description 4
- 239000002054 inoculum Substances 0.000 description 4
- 239000002105 nanoparticle Substances 0.000 description 4
- 235000019319 peptone Nutrition 0.000 description 4
- 238000005498 polishing Methods 0.000 description 4
- 230000001954 sterilising effect Effects 0.000 description 4
- 241001646719 Escherichia coli O157:H7 Species 0.000 description 3
- 239000012141 concentrate Substances 0.000 description 3
- 229920001817 Agar Polymers 0.000 description 2
- 239000006142 Luria-Bertani Agar Substances 0.000 description 2
- 239000006137 Luria-Bertani broth Substances 0.000 description 2
- 239000008272 agar Substances 0.000 description 2
- 239000011248 coating agent Substances 0.000 description 2
- 241000193738 Bacillus anthracis Species 0.000 description 1
- VKJGBAJNNALVAV-UHFFFAOYSA-M Berberine chloride (TN) Chemical compound [Cl-].C1=C2CC[N+]3=CC4=C(OC)C(OC)=CC=C4C=C3C2=CC2=C1OCO2 VKJGBAJNNALVAV-UHFFFAOYSA-M 0.000 description 1
- 241000218202 Coptis Species 0.000 description 1
- 241000037803 Coptis deltoidea Species 0.000 description 1
- 235000002991 Coptis groenlandica Nutrition 0.000 description 1
- 206010012735 Diarrhoea Diseases 0.000 description 1
- 241000700721 Hepatitis B virus Species 0.000 description 1
- 240000002853 Nelumbo nucifera Species 0.000 description 1
- 235000006508 Nelumbo nucifera Nutrition 0.000 description 1
- 235000006510 Nelumbo pentapetala Nutrition 0.000 description 1
- 241000589517 Pseudomonas aeruginosa Species 0.000 description 1
- 241000218201 Ranunculaceae Species 0.000 description 1
- 241000191967 Staphylococcus aureus Species 0.000 description 1
- 241000194017 Streptococcus Species 0.000 description 1
- 241000193998 Streptococcus pneumoniae Species 0.000 description 1
- 230000003110 anti-inflammatory effect Effects 0.000 description 1
- 230000000259 anti-tumor effect Effects 0.000 description 1
- 230000006793 arrhythmia Effects 0.000 description 1
- 206010003119 arrhythmia Diseases 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 208000001848 dysentery Diseases 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 210000000232 gallbladder Anatomy 0.000 description 1
- 230000005802 health problem Effects 0.000 description 1
- 230000002949 hemolytic effect Effects 0.000 description 1
- 230000002519 immonomodulatory effect Effects 0.000 description 1
- 239000004615 ingredient Substances 0.000 description 1
- 210000002429 large intestine Anatomy 0.000 description 1
- 230000001717 pathogenic effect Effects 0.000 description 1
- 229920000642 polymer Polymers 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 229940031000 streptococcus pneumoniae Drugs 0.000 description 1
- 230000002195 synergetic effect Effects 0.000 description 1
- 241000712461 unidentified influenza virus Species 0.000 description 1
Classifications
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
The invention discloses a kind of suppression micrococcus luteus Rhizoma Coptidis extract and nano-silver bacteriostatic composition, belong to the field of Chinese medicines.In bacteria inhibiting composition, Rhizoma Coptidis extract and water-soluble nano money kind medicine share suppressing micrococcus luteus to play collaborative effect.A kind of Rhizoma Coptidis extract of the present invention and nano-silver bacteriostatic composition are compared with prior art, solve the problem of Rhizoma Coptidis 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
Technical field
The invention belongs to the field of Chinese medicines, particularly relate to a kind of Traditional Chinese medicine bacteriostatic composition.
Background technology
Coptis perennial herb, rhizome is yellow, and normal branch, the most fibrous root of close life is the Rhizoma Coptidis of Ranunculaceae, the dry rhizome of Coptis deltoidea C.Y.Cheng et Hsiao or cloud lotus.It is mainly distributed in Sichuan, Guizhou, Hunan, Hubei, Southern Shaanxi, has heat clearing and damp drying, effect of eliminating fire and detoxication.Berberine hydrochloride (berberine) is its main effective ingredient.Modern pharmacology experimentation shows that Rhizoma Coptidis has resisting pathogenic microbes effect, simultaneously all inhibited to staphylococcus aureus, large intestine Erichsen bacterium, Hemolytic streptococcus, streptococcus pneumoniae, Pseudomonas aeruginosa, meningococcus, dysentery bacterium, anthrax bacillus, influenza virus, hepatitis B virus etc.In addition, Rhizoma Coptidis also has the effects such as blood sugar lowering, antitumor, diarrhea, function of gallbladder promoting, arrhythmia, antiinflammatory and immunomodulating.Rhizoma Coptidis is worth because it has higher medicine and health care, extensively causes people and pays close attention to.
Chinese medicine preparation concentrated solution makes oral liquid or bacteria inhibiting composition, coating agent etc. are once large with dosage, and life-time service affects health.
Summary of the invention
For Rhizoma Coptidis bacteria inhibiting composition ampoule, large and life-time service affects human health problems, the present inventor, through great many of experiments, finds that nanometer silver can effectively strengthen Rhizoma Coptidis extract bacteriostasis property, reduces the consumption of Rhizoma Coptidis antibacterial, and adopt the bacteria inhibiting composition of two kinds of low dosages, more safe.The object of the present invention is to provide a kind of Rhizoma Coptidis nano-silver bacteriostatic composition, reduce Rhizoma Coptidis antibacterial use dosage once and reduce medicine to the toxicity of human body.
The invention provides following technical scheme:
A kind of bacteria inhibiting composition, containing Rhizoma Coptidis extract, water-soluble nano silver;
Rhizoma Coptidis extract preparation comprises the steps: to get dry medical material Rhizoma Coptidis, and amount of water is 9 times of Rhizoma Coptidis weight, extracts 4h, then obtain extracting solution through concentrating under reduced pressure through 95 DEG C of water-baths; Gained extracting solution is through 0.22 μm of membrane filtration.Gained extracting solution carries out standard quantitative through high performance liquid chromatography to berberine.
Suppress Escherichia coli O 157: during H7, the volume ratio of Rhizoma Coptidis extract, water-soluble nano silver is 26.6 ~ 30:1.When suppressing micrococcus luteus, the volume ratio of Rhizoma Coptidis extract, water-soluble nano silver is 1 ~ 1.2:1.When calculating volume, in extracting solution, the content of berberine is: 28mg/mL, water-soluble nano silver concentration is 10mg/mL.
Water-soluble nano silver grain diameter is 3 ~ 10nm, the coated amphiphilic polymers of nanometer silver appearance.
Water-soluble nano silver particle diameter is 3 ~ 10nm, appearance is carboxyl, prepare 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 water-soluble nano Argent grain prepared according to this method and Rhizoma Coptidis extract have good synergetic antibacterial effect.
The invention still further relates to above-mentioned bacteria inhibiting composition at suppression Escherichia coli O 157: the application in H7.
The invention still further relates to above-mentioned bacteria inhibiting composition and suppress the application in micrococcus luteus.
The invention has the beneficial effects as follows: the application of water-soluble nano silver of the present invention can effectively strengthen Rhizoma Coptidis bacteriostasis property, reduce the consumption of Rhizoma Coptidis antibacterial, more unexpected is that share of two kinds of medicines plays synergism to suppression Escherichia coli O 157: H7 and micrococcus luteus, this bacteria inhibiting composition adopts the antibacterial substance of two kinds of low dosages simultaneously, relative to the Rhizoma Coptidis or the water-soluble nano silver that use high dose, more safe, can as external coating agent etc. safely and reliably.
Accompanying drawing explanation
Fig. 1 Rhizoma Coptidis extract and the ratio of water-soluble nano silver are the collaborative antibacterial of 30:1
Fig. 1 a2 μ L nanometer silver is to Escherichia coli O 157: the suppression result of H7, and Fig. 1 b60 μ L Rhizoma Coptidis extract is to Escherichia coli O 157: the suppression result of H7, and Fig. 1 c30 μ L Rhizoma Coptidis extract and 1 μ L nanometer silver are to Escherichia coli O 157: the suppression result of H7.
Fig. 2 Rhizoma Coptidis extract and the ratio of water-soluble nano silver are the collaborative antibacterial of 26.6:1
What Fig. 2 a represented is 1.5 μ L nanometer silvers are to Escherichia coli O 157: the antibacterial result of H7, what Fig. 2 c represented is 40 μ L Rhizoma Coptidis extract are to Escherichia coli O 157: the antibacterial result of H7, and what Fig. 2 c represented is 0.75 μ L nanometer silver, 20 μ L Rhizoma Coptidis extract mixture are to Escherichia coli O 157: the collaborative antibacterial result of H7.
Fig. 3 Rhizoma Coptidis extract and the ratio of water-soluble nano silver be 1 collaborative antibacterial
What Fig. 3 a represented is that 16 μ L nanometer silvers are to the antibacterial result of micrococcus luteus, what Fig. 3 b represented is 16 μ L Rhizoma Coptidis extract are to the antibacterial result of micrococcus luteus, and what Fig. 3 c represented is that 8 μ L nanometer silvers, 8 μ L Rhizoma Coptidis extract are to the collaborative antibacterial result of micrococcus luteus.
Fig. 4 Rhizoma Coptidis extract and the ratio of water-soluble nano silver are the collaborative antibacterial of 5:8
What Fig. 4 a represented is that 16 μ L nanometer silvers are to the antibacterial result of micrococcus luteus, what Fig. 4 b represented is 10 μ L Rhizoma Coptidis extract are to the antibacterial result of micrococcus luteus, and what Fig. 4 c represented is 8 μ L nanometer silvers and 5 μ L Rhizoma Coptidis extract are to the collaborative antibacterial result of micrococcus luteus.
Detailed description of the invention
Embodiment 1
1, the preparation process of Rhizoma Coptidis extract: take dry medical material Rhizoma Coptidis 50g, amount of water is 450mL, 4h is extracted at 95 DEG C, concentrate through vacuum rotating concentrating instrument again and obtain extracting solution, after 0.22 μm of membrane filtration, with the content that high performance liquid chromatography records berberine in extracting solution be again finally: 28mg/mL.
2, water-soluble silver nano-particle preparation:
Get 22.8g tetradecanoic acid to 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, and 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.
3, Rhizoma Coptidis extract and water-soluble nano silver work in coordination with bacteriostasis.
The cultivation of a antibacterial
Single bacterium colony of the Escherichia coli O 157 on picking LB agar culture medium: H7, in 10mLLB broth bouillon, is placed in 37 DEG C of incubators and cultivates 16h, then is inoculated in the LB broth bouillon of 5mL by the inoculum concentration of 1%, after 37 DEG C of cultivation 4h.Get the above-mentioned bacterium liquid of 1mL with 0.1% peptone water serial dilution three gradients, final bacterium liquid is approximately 10
6cFU/mL.
B water-soluble nano silver and the Rhizoma Coptidis extract synergism on bacteriostasis property
Get the above-mentioned bacterium liquid diluted of 1mL respectively in the centrifuge tube of 1.5mL sterilizing, in No. 1 centrifuge tube, adding 60 μ L Rhizoma Coptidis extract respectively, (content of berberine is 28mg/mL, lower same), in No. 2 centrifuge tubes, add 2 μ L concentration is the water-soluble nano silver of 10mg/mL, in No. 3 centrifuge tubes, add 30 μ L Rhizoma Coptidis extract and 1 μ L concentration is 10mg/mL water-soluble nano silver, do not add any above-mentioned antibacterial.For ensureing that often pipe liquid final volume is equal, residual quantity with 0.1% peptone water polishing.Be placed in 37 DEG C and cultivate 2h.Often parallel three times of group experiment.
C plate count
By liquid PBS to be measured for experimental group serial dilution two gradients, 10
0, 10
-1, 10
-2each taking-up 200 μ L is evenly coated with respectively
Cloth is on LB solid plate; Blank group serial dilution four gradients, from 10
-2, 10
-3, 10
-4three gradients are all coated on respectively
On LB solid plate.After flat board dries up, be inverted for 37 DEG C and cultivate 12h, count after growing bacterium colony, with 30-300 colony-forming units (CFU) for effective count range.Every milliliter of original bacteria liquid viable count=plate count * extension rate * 5
Experimental result is as follows:
| Antibacterial | The clump count that can count after adding antibacterial |
| Be the carboxylated nanometer silver of 10 mg/mL containing 2 μ L concentration | 390 CFU/mL |
| 60 μ L Rhizoma Coptidis extract | 1850 CFU/mL |
| Be the carboxylated nanometer silver of 10 mg/mL and 30 μ L Rhizoma Coptidis extract containing 1 μ L concentration | 0 CFU/mL |
As shown in Figure 1, Fig. 1 a2 μ L nanometer silver is to Escherichia coli O 157: the suppression result of H7, and Fig. 1 b60 μ L Rhizoma Coptidis extract is to Escherichia coli O 157: the suppression result of H7, and Fig. 1 c30 μ L Rhizoma Coptidis extract and 1 μ L nanometer silver are to Escherichia coli O 157: the suppression result of H7.
Experiment shows: bacteria inhibiting composition provided by the invention has the effect reducing Rhizoma Coptidis antibacterial ampoule, and share of two kinds of medicines plays collaborative effect to suppression Escherichia coli O 157: H7.
Embodiment 2
1, the preparation process of Rhizoma Coptidis extract: take Rhizoma Coptidis 50g, amount of water is 450mL, 4h is extracted at 95 DEG C, concentrate through vacuum rotating concentrating instrument again and obtain extracting solution, after 0.22 μm of membrane filtration, with the content that high performance liquid chromatography records berberine in extracting solution be again finally: 28mg/mL.
2, water-soluble silver nano-particle preparation process.
With embodiment 1
3, Rhizoma Coptidis extract and water-soluble nano silver work in coordination with bacteriostasis
The cultivation of a antibacterial
Single bacterium colony of the Escherichia coli O 157 on picking LB agar culture medium: H7, in 10mLLB broth bouillon, is placed in 37 DEG C of incubators and cultivates 16h, then is inoculated in the LB broth bouillon of 5mL by the inoculum concentration of 1%, after 37 DEG C of cultivation 4h.Get the above-mentioned bacterium liquid of 1mL with 0.1% peptone water serial dilution three gradients, final bacterium liquid is approximately 10
6cFU/mL.
B water-soluble nano silver and the Rhizoma Coptidis extract synergism on bacteriostasis property
Get the above-mentioned bacterium liquid diluted of 1mL respectively in the centrifuge tube of 1.5mL sterilizing, in No. 1 centrifuge tube, adding the Rhizoma Coptidis extract of 40 μ L respectively, (content of berberine is: 28mg/mL), in No. 2 centrifuge tubes, add 1.5 μ L concentration is the water-soluble nano silver of 10mg/mL, in No. 3 centrifuge tubes, add 20 μ L Rhizoma Coptidis extract and 0.75 μ L concentration is 10mg/mL water-soluble nano silver, do not add any above-mentioned antibacterial.For ensureing that often pipe liquid final volume is equal, residual quantity with 0.1% peptone water polishing.Be placed in 37 DEG C and cultivate 2h.Often parallel three times of group experiment.
C plate count
By liquid PBS to be measured for experimental group serial dilution two gradients, 10
0, 10
-1, 10
-2each taking-up 200 μ L is evenly coated with respectively
Cloth is on LB solid plate; Blank group serial dilution four gradients, from 10
-2, 10
-3, 10
-4three gradients are all coated on respectively
On LB solid plate.After flat board dries up, be inverted for 37 DEG C and cultivate 12h, count after growing bacterium colony, with 30-300 colony-forming units (CFU) for effective count range.Every milliliter of original bacteria liquid viable count=plate count * extension rate * 5
Experimental result is as follows:
| Antibacterial | The clump count that can count after adding antibacterial |
| Be the carboxylated nanometer silver of 10 mg/mL containing 1.5 μ L concentration | 510 CFU/mL |
| 40 μ L Rhizoma Coptidis extract | 2000 CFU/mL |
| Be the carboxylated nanometer silver of 10 mg/mL and 20 μ L Rhizoma Coptidis extract containing 0.75 μ L concentration | 0 CFU/mL |
As shown in Figure 2, what Fig. 2 a represented is 1.5 μ L nanometer silvers are to Escherichia coli O 157: the antibacterial result of H7, what Fig. 2 b represented is 40 μ L Rhizoma Coptidis extract are to Escherichia coli O 157: the antibacterial result of H7, and what Fig. 2 c represented is 0.75 μ L nanometer silver, 20 μ L Rhizoma Coptidis extract mixture are to Escherichia coli O 157: the collaborative antibacterial result of H7.
Experiment shows: bacteria inhibiting composition provided by the invention has the effect reducing Rhizoma Coptidis antibacterial ampoule, and share of two kinds of medicines plays collaborative effect to suppression Escherichia coli O 157: H7.
Embodiment 3
1, the preparation process of Rhizoma Coptidis extract: take Rhizoma Coptidis 50g, amount of water is 450mL, at 95 DEG C, extract 4h, then obtains extracting solution through vacuum rotating concentrating instrument is concentrated, after 0.22 μm of membrane filtration, with the content that high performance liquid chromatography records berberine in extracting solution be again finally: 28mg/mL
2, water-soluble silver nano-particle preparation:
With embodiment 1
3, Rhizoma Coptidis extract and water-soluble nano silver work in coordination with bacteriostasis
The cultivation of a antibacterial
Micrococcus luteus list bacterium colony on picking beef extract-peptone agar culture medium is in 5mL beef extract-peptone fluid medium, be placed in 30 DEG C of incubators and cultivate 24h, be inoculated in the beef extract-peptone fluid medium of 5mL by the inoculum concentration of 1% again, after 30 DEG C of training sample 12h.Get the above-mentioned bacterium liquid of 1mL serial dilution three gradients in the mixed liquor mixed in 4:1 ratio with PBS and fluid medium, final bacterium liquid is approximately 10
6cFU/mL.
B water-soluble nano silver and the Rhizoma Coptidis extract synergism on bacteriostasis property
Get the above-mentioned bacterium liquid diluted of 1mL respectively in the centrifuge tube of 1.5mL sterilizing, in No. 1 centrifuge tube, adding 16 μ L Rhizoma Coptidis extract respectively, (content of berberine is: 28mg/mL), in No. 2 centrifuge tubes, add 16 μ L concentration is the water-soluble nano silver of 10mg/mL, in No. 3 centrifuge tubes, add 8 μ L Rhizoma Coptidis extract and 8 μ L concentration are 10mg/mL water-soluble nano silver, do not add any above-mentioned antibacterial.For ensureing that often pipe liquid final volume is equal, the mixed liquor polishing that residual quantity PBS mixes in 4:1 ratio with fluid medium.Be placed in 30 DEG C and cultivate 6h.Often parallel three times of group experiment.
C plate count
By liquid PBS to be measured for experimental group serial dilution two gradients, 10
-1, 10
-2, 10
-3each taking-up 100 μ L is evenly coated with respectively
Cloth is on beef extract-peptone solid plate; Blank group serial dilution four gradients, from 10
-2, 10
-3, 10
-4three gradients are uniformly coated on beef extract-peptone solid plate respectively.After flat board dries up, be inverted for 30 DEG C and cultivate 48h, count after growing bacterium colony, with 20-200 colony-forming units (CFU) for effective count range.Every milliliter of original bacteria liquid viable count=plate count * extension rate * 10
| Antibacterial | The clump count that can count after adding antibacterial |
| Be the carboxylated nanometer silver of 10 mg/mL containing 16 μ L concentration | 3.1 x10 5 CFU/mL |
| 16 μ L Rhizoma Coptidis extract | 200 CFU/mL |
| Be 10 mg/ml carboxylated nanometer silver+8 μ L Rhizoma Coptidis extract containing 8 μ L concentration | 0 CFU/mL |
As shown in Figure 3, what Fig. 3 a represented is that 16 μ L nanometer silvers are to the antibacterial result of micrococcus luteus, what Fig. 3 b represented is 16 μ L Rhizoma Coptidis extract are to the antibacterial result of micrococcus luteus, and what Fig. 3 c represented is that 8 μ L nanometer silvers, 8 μ L Rhizoma Coptidis extract are to the collaborative antibacterial result of micrococcus luteus.
Experiment shows: bacteria inhibiting composition provided by the invention has the effect reducing Rhizoma Coptidis antibacterial ampoule, and share of two kinds of medicines plays collaborative effect to suppression micrococcus luteus.
Embodiment 4
1, the preparation process of Rhizoma Coptidis extract: take Rhizoma Coptidis 50g, amount of water is 450mL, 4h is extracted at 95 DEG C, concentrate through vacuum rotating concentrating instrument again and obtain extracting solution, after 0.22 μm of membrane filtration, with the content that high performance liquid chromatography records berberine in extracting solution be again finally: 28mg/mL.
2, water-soluble silver nano-particle preparation:
With embodiment 1.
3, Rhizoma Coptidis extract and water-soluble nano silver work in coordination with bacteriostasis
The cultivation of a antibacterial
Micrococcus luteus list bacterium colony on picking beef extract-peptone agar culture medium is in 5mL beef extract-peptone fluid medium, be placed in 30 DEG C of culture medium and cultivate 24h, be inoculated in the beef extract-peptone fluid medium of 5mL by the inoculum concentration of 1% again, after 30 DEG C of training sample 12h.Get the above-mentioned bacterium liquid of 1mL serial dilution three gradients in the mixed liquor mixed in 4:1 ratio with PBS and fluid medium, final bacterium liquid is approximately 10
6cFU/mL.
B water-soluble nano silver and the Rhizoma Coptidis extract synergism on bacteriostasis property
Get the above-mentioned bacterium liquid diluted of 1mL respectively in the centrifuge tube of 1.5mL sterilizing, in No. 1 centrifuge tube, adding 10 μ L Rhizoma Coptidis extract respectively, (content of berberine is: 28mg/mL), in No. 2 centrifuge tubes, add 8 μ L concentration is the water-soluble nano silver of 10mg/mL, in No. 3 centrifuge tubes, add the water-soluble nano silver water-soluble nano silver that 5 μ L Rhizoma Coptidis extract and 4 μ L concentration are 10mg/mL, do not add any above-mentioned antibacterial.For ensureing that often pipe liquid final volume is equal, the mixed liquor polishing that residual quantity PBS mixes in 4:1 ratio with fluid medium.Be placed in 30 DEG C and cultivate 6h.Often parallel three times of group experiment.
C plate count
By liquid PBS to be measured for experimental group serial dilution two gradients, 10
-1, 10
-2, 10
-3each taking-up 100 μ L is evenly coated with respectively
Cloth is on beef extract-peptone solid plate; Blank group serial dilution four gradients, from 10
-2, 10
-3, 10
-4three gradients are uniformly coated on beef extract-peptone solid plate respectively.After flat board dries up, be inverted for 30 DEG C and cultivate 48h, count after growing bacterium colony, with 20-200 colony-forming units (CFU) for effective count range.Every milliliter of original bacteria liquid viable count=plate count * extension rate * 10
| Antibacterial | The clump count that can count after adding antibacterial |
| Be the carboxylated nanometer silver of 10 mg/mL containing 16 μ L concentration | 4.5 x10 5 CFU/mL |
| 10 μ L Rhizoma Coptidis extract | 1000 CFU/mL |
| Be the carboxylated nanometer silver of 10 mg/mL and 5 μ L Rhizoma Coptidis extract containing 8 μ L concentration | 100 CFU/mL |
As shown in Figure 4, what Fig. 4 a represented is that 16 μ L nanometer silvers are to the antibacterial result of micrococcus luteus, what Fig. 4 b represented is 10 μ L Rhizoma Coptidis extract are to the antibacterial result of micrococcus luteus, and what Fig. 4 c represented is 8 μ L nanometer silvers and 5 μ L Rhizoma Coptidis extract are to the collaborative antibacterial result of micrococcus luteus.
Experiment shows: bacteria inhibiting composition provided by the invention has the effect reducing Rhizoma Coptidis antibacterial ampoule, and share of two kinds of medicines plays collaborative effect to suppression micrococcus luteus.
Claims (2)
1. suppress Rhizoma Coptidis extract and the nano-silver bacteriostatic composition of micrococcus luteus, it is characterized in that being made up of Rhizoma Coptidis extract, water-soluble nano silver; Described Rhizoma Coptidis extract preparation comprises the steps: to get dry medical material Rhizoma Coptidis, and amount of water is 9 times of Rhizoma Coptidis weight, and extract 4h through 95 DEG C of water-baths, then obtain extracting solution through concentrating under reduced pressure, gained extracting solution is through 0.22 μm of membrane filtration; 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 Rhizoma Coptidis extract, water-soluble nano silver is 1 ~ 1.2:1; In extracting solution, the content of berberine is 28mg/mL, and water-soluble nano silver concentration is 10mg/mL.
2. bacteria inhibiting composition suppresses the application in micrococcus luteus medicine in preparation as claimed in claim 1.
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