KR101587811B1 - A composition containing cryptotanshinone as a active ingredient for the inhibition of mrsa and vrsa - Google Patents

Abstract

The present invention relates to a composition for antimicrobial use of MRSA and / or VRSA containing cryptoxanone as an active ingredient.
The present invention relates to a composition for antimicrobial use of MRSA and / or VRSA containing as an active ingredient a mixture of cryptotansinone and a conventional antimicrobial substance.
In particular, it has an effect of causing a synergistic effect by containing a mixture of cryptotansinone and an existing antimicrobial substance as an active ingredient.
In addition, MRSA or VRSA is completely killed after 3 ~ 6 hours of treatment with a mixture of cryptotansinone 1/2 MIC value and a conventional antimicrobial substance 1/2 MIC value.
The present invention relates to a health functional food for antimicrobial use of MRSA or VRSA containing cryptoxanone as an active ingredient.

Description

TECHNICAL FIELD The present invention relates to a composition for antimicrobial use of MRSA and VRSA containing cryptoxanthinone as an active ingredient,

The present invention relates to a composition for antimicrobial use of MRSA and / or VRSA containing cryptoxanone as an active ingredient.

In particular, cryptoxanthin has an efficacy that corresponds to a minimum inhibitory concentration (MIC) value of 0.5 to 64 ug / ml and a minimum bactericidal concentration (MBC) value of 2 to 128 ug / ml for MRSA and / or VRSA have.

The present invention relates to a composition for antimicrobial use of MRSA and / or VRSA containing as an active ingredient a mixture of cryptotansinone and a conventional antimicrobial substance.

In particular, it has an effect of causing a synergistic effect by containing a mixture of cryptotansinone and an existing antimicrobial substance as an active ingredient.

In addition, MRSA or VRSA is completely killed after 3 ~ 6 hours of treatment with a mixture of cryptotansinone 1/2 MIC value and a conventional antimicrobial substance 1/2 MIC value.

Staphylococcus bacteria are widely distributed in nature and are resistant to the environment and are capable of survival for a long time without parasitic organisms. In 1878 Koch first observed the morphology of Staphylococcus bacteria on a microscope.

Staphylococcus aureus is a It is a species belonging to the genus Staphylococcus. It is normally present in the skin, nasal cavity, upper urinary tract, urinary tract, digestive system of healthy person, and also in the surrounding environment such as air, floor and house. Even in the oral cavity, such as the mucous membranes of the throat.

They do not cause problems when their immunity is normal, but they can be opportunistic infections through wound or respiratory tract of the skin in a state of decreased immunity such as surgery, burn, immunodeficiency disease infection. These are Gram-positive strains that cause local and systemic infections and are the leading cause of infectious diseases that account for more than 80% of the infectious infection. In addition, oral antibiotics such as sore throat and tonsillitis cause infectious disease.

In the early days when β-lactam antibiotics such as penicillin were developed and used, β-lactam antibiotics were highly susceptible to Staphylococcus aureus, which facilitated the treatment. However, β-lactam antibiotics Has become more tolerant to starfycoccus aureus. Staphylococcus aureus possesses resistance as a principle for producing [beta] -lactamase, an enzyme that inactivates [beta] -lactam antibiotics.

Methicillin, which is not inactivated by β-lactamase, has been developed and used to treat Staphylococcus aureus infection. However, in the early 1960s, methicillin-resistant Staphylococcus aureus (penicillin- MRSA) was found. MRSA is resistant to β-lactam antibiotics as well as other antibiotics.

MRSA can form biofilms in implants, medical supplies or medical devices implanted in a patient. Biofilms that are formed once can not be easily removed and can cause serious pathogenic infections such as sepsis. .

In addition, MRSA can produce organic acids by metabolizing saccharides through the process. This saccharide metabolism process is an important energy acquisition mechanism necessary for the survival of bacteria. It is known that organic acids produced by MRSA can promote biofilm formation by lowering the pH.

On the other hand, vancomycin is a therapeutic agent developed to replace methicillin. Streptomyces It is the antibiotic that has the most powerful antibiotic developed so far. There are serious side effects such as antibiotics that MRSA only responds to, acuity disorder, kidney disorder, liver failure, bacterial diabetes and anaphylactic shock.

Vancomycin-resistant Staphylococcus, which does not respond to vancomycin in Japan in 1996 aureus (VRSA) was found. VRSA possesses characteristics similar to MRSA to form biofilm, and can cause sepsis and the like, and it is impossible to inhibit activity with antibiotics developed so far.

As mentioned above, MRSA is a bacterium that does not easily inhibit its activity because it has characteristics of showing multiple drug resistance and biofilm formation. VRSA is a bacterium which can not be inhibited by any antibiotics developed so far . Therefore, the development of new drugs that can treat infections of MRSA and VRSA is urgently required.

Accordingly, the Applicant has found a composition which can inhibit the activity of MRSA and / or VRSA effectively by using a natural-derived substance and having no side effects and no resistance.

In particular, a composition for antimicrobial use of MRSA and / or VRSA containing cryptoxanthinone derived from Danshen as an active ingredient has been disclosed.

Also disclosed is a composition for antimicrobial use of MRSA and / or VRSA containing as an active ingredient a mixed composition of cryptoxanthinone and / or existing antimicrobial substance.

Hereinafter, the present invention will be described in brief with reference to a plant derived from cryptotansinone and cryptotansinone, which are effective ingredients of the present invention.

Cryptotanshinone (C ₁₉ H ₂₀ O ₃ ) belongs to the Tanshinone family, and is one of the major active ingredients of the ginseng extract. (COX-2), which inhibits the function of inflammation, is effective in preventing inflammation, antioxidation, antimicrobial, anti-angiogenesis, anti-mutation, anti-platelet aggregation, . Coronary artery related diseases such as hyperlipidemia, ischemic diseases, chronic kidney disease and chronic hepatitis are effective.

Salvia miltiorrhiza Bunge) is a perennial plant belonging to the family Lamiaceae, and medicinal sites are root and root. It is distributed in China, Gyeongsangbuk-do, and Gangwon-do in Korea, China, Japan and Korea. It grows on the hill of the mountains. The height of the stem is 40 ~ 80cm and the whole is hairy. The roots are reddish, with a characteristic odor and a bitter taste. Leaves are opposite to each other and petiole is long. The tip of the leaf is pointed and has dull serrations on the edge. Flowers are purple, 2 ~ 2.5cm long, run in stratum at the end of stem, and bloom in June ~ August. The petals are 3 pronged, with serrate on the edge. Fruits are 2-3 small nuts, egg-shaped and plain. The flowering season is from June to August. It grows mainly in Korea.

Tanshinone I, II (tanshinone I, II), dihydrotanshinone, cryptotanshinone, methyl tanshinone, methylene, tanshiquinone, And beta-sitosterol. ≪ / RTI >

Dansam is effective against cardiovascular diseases, hepatitis, gynecological diseases, diabetes, and chronic kidney diseases.

Meanwhile, Korean Patent Laid-Open Publication No. 10-2010-0114606 discloses a mandramic composition for antimicrobial use of MRSA, and Korean Patent Publication No. 10-0967541 discloses a composition for antibacterial activity against MRSA and / or VRSA Desc / Clms Page number 2 >

The materials of these patent documents are different from cryptotansinone or danshen, and there is no mention about the antibacterial matters of VRSA.

In addition, the prior art patents related to the inhibition of MRSA and / or VRSA activity of Dansamp are described as active ingredients of natural food preservatives in Korean Patent Laid-Open Publication No. 10-2012-0073389.

However, the above-mentioned patent document only shows antimicrobial activity against Staphylococcus sp. Bacterium using the extract of Panax ginseng. Since MRSA and VRSA are resistant to antibiotics and have different characteristics from those of Staphylococcus bacteria, The above-mentioned patent documents do not describe the matters concerning the inhibition of the activity of MRSA and / or VRSA.

Korean Patent Publication No. 10-2010-0114606 (October 26, 2010) Korean Registered Patent No. 10-0967541 (June 25, 2010) Korean Patent Publication No. 10-2012-0073389 (Jul. 05, 2012)

Ge Young Young. Antimicrobial Activity and Resistance Gene Modulation of Antibiotic Resistant Strain (MRSA) from Salvamilthorrhiza Bunge Extracts. Master Thesis. Graduate School of Keimyung University. 2006.12

It is an object of the present invention to provide a composition for antimicrobial use of MRSA and / or VRSA containing a naturally occurring substance as an active ingredient.

It is an object of the present invention to provide a composition for antimicrobial use of MRSA and / or VRSA containing cryptosanicin as an active ingredient mainly derived from persimmon.

It is an object of the present invention to provide a pharmaceutical composition containing cryptoxanthinone as an active ingredient and having a minimum inhibitory concentration (MIC) value of 0.5 to 64 ug / ml and a minimum sterilization concentration (MBC) value of 2 to 128 for MRSA and / or VRSA And to provide a composition for antimicrobial use of MRSA and / or VRSA having the corresponding efficacy.

It is an object of the present invention to provide a composition for antimicrobial use of MRSA and / or VRSA, which has an effect of causing a synergistic effect by containing a mixture of cryptotansinone and a conventional antimicrobial substance as an active ingredient.

It is also an object of the present invention to provide a method for the treatment of MRSA and / or VRSA, which has the complete killing effect of MRSA or VRSA after 3 to 6 hours of treatment with a mixture of cryptoxanthin 1/2 MIC value and 1/2 antimicrobial MIC value, Or a composition for antibiosis of VRSA.

The present invention aims at solving the technical problem by providing a composition for antimicrobial use of MRSA and / or VRSA containing a substance derived from nature as an active ingredient.

It is an object of the present invention to solve the technical problem by providing a composition for antimicrobial use of MRSA and / or VRSA containing cryptosanicin as an active ingredient mainly derived from Danshen.

It is an object of the present invention to provide a pharmaceutical composition containing cryptoxanthinone as an active ingredient and having a minimum inhibitory concentration (MIC) value of 0.5 to 64 ug / ml and a minimum sterilization concentration (MBC) value of 2 to 128 for MRSA and / or VRSA To provide a composition for antimicrobial use of MRSA and / or VRSA, which has the corresponding efficacy.

It is an object of the present invention to provide a composition for antimicrobial use of MRSA and / or VRSA, which has an effect of causing a synergistic effect by containing a mixture of cryptotansinone and an existing antimicrobial substance as an active ingredient, thereby solving the technical problem I want to.

It is also an object of the present invention to provide a method for the treatment of MRSA and / or VRSA, which has the complete killing effect of MRSA or VRSA after 3 to 6 hours of treatment with a mixture of cryptoxanthin 1/2 MIC value and 1/2 antimicrobial MIC value, Or a composition for antibiosis of VRSA.

The present invention has an effect of antibacterial activity of MRSA and / or VRSA by containing a natural-derived substance as an active ingredient.

The present invention has antimicrobial activity against MRSA and / or VRSA containing cryptoxanthin as an active ingredient mainly derived from persimmon.

The present invention relates to a pharmaceutical composition comprising cryptoxanthinone as an active ingredient and having a minimum inhibitory concentration (MIC) value for MRSA and / or VRSA of 0.5 to 64 ug / ml and a minimum sterilization concentration (MBC) value of 2 to 128 It has efficacy.

The present invention has an effect of causing a synergistic effect by containing a mixture of cryptotansinone and an existing antimicrobial substance as an active ingredient.

The present invention also shows that MRSA or VRSA is completely killed after 3 to 6 hours of treatment with a mixture of cryptoxanthin 1/2 MIC and 1/2 antimicrobial MIC.

Figure 1 shows the results of treatment of cryptotansinone, cryptoxanthin and ampicillin, cryptotansonone and oxacillin, cryptoxanone and vancomycin in MSSA 25923, MRSA 33591, MSSA 1 and MSSA 2, and VRSA 1 and VRSA 2 FIG. 4 is a graph showing the antimicrobial effect of the composition according to the present invention. FIG.
FIG. 2 shows the results of treatment of cryptotansinone, cryptoxanthin and ampicillin, cryptotansonone and oxacillin, cryptoxanthone and vancomycin in MRSA 1, MRSA 2, MRSA 3, MRSA 4, MRSA 5 and MRSA 6 FIG. 4 is a graph showing the antimicrobial effect of the composition according to the present invention. FIG.
Figure 3 shows the results of treatment of cryptotansinone, cryptotansinone and ampicillin, cryptotansinone and oxacillin, cryptoxanthone and vancomycin in MRSA 7, MRSA 8, MRSA 9, MRSA 10, MRSA 11 and MRSA 12, FIG. 4 is a graph showing the antimicrobial effect of the composition according to the present invention. FIG.
Figure 4 shows the antimicrobial effect patterns of MRSA 13, MRSA 14, MRSA 15 and MRSA 16 after treatment with cryptotansinone, cryptoxanthin and ampicillin, cryptotansinone and oxacillin, cryptoxanthinone and vancomycin, respectively Fig.

The terms and words used in the present specification and claims should not be construed as limited to ordinary or dictionary terms and the inventor may properly define the concept of the term in order to best describe its invention It should be construed as meaning and concept consistent with the technical idea of the present invention.

Therefore, the embodiments described in the present specification, the reference examples, and the drawings are merely the most preferred examples of the present invention, and not all of the technical ideas of the present invention are described. Therefore, It should be understood that various equivalents and modifications may be present.

Example  One. MRSA  And / or VRSA (1) < / RTI >

The root or root of Salvia miltiorrhiza Bunge is extracted or fractionated with water, ethanol, methanol or other organic solvent, and then cryptotansinone is isolated and identified. Depending on the type of extraction solvent, the extraction temperature, the extraction time, the amount of solvent, and the method of treating the residual components are treated differently.

Concentrate the isolated cryptosaccharide to a concentration of at least 2 - 128 ug / ml and then use it as an active ingredient. This indicates that cryptoxanthin has an efficacy that corresponds to a minimum inhibitory concentration (MIC) value of 2 to 64 ug / ml and a minimum bactericidal concentration (MBC) value of 4 to 128 ug / ml for MRSA or VRSA Based.

Or cryptoxanthin concentration is at least 0.5 to 128 ug / ml. This is based on the fact that 1/2 MIC values for MRSA and / or VRSA of cryptoxanthin have an efficacy corresponding to 0.5 to 8 ug / ml.

Example  2. MRSA  And / or VRSA Composition for antimicrobial (2)

(1) The root or root of Salvia miltiorrhiza Bunge is extracted or fractionated using water, ethanol, methanol or other organic solvent, and then cryptotansinone is isolated and identified. Depending on the type of extraction solvent, the extraction temperature, the extraction time, the amount of solvent, and the method of treating the residual components are treated differently.

Concentrate the isolated cryptosaccharide to a concentration of at least 2 to 64 ug / ml.

Or cryptoxanthin concentration may be at least 0.5 to 64 ug / ml. This is based on the fact that 1/2 MIC values for cryptotansic MRSA or VRSA antibiotics have an efficacy corresponding to 0.5 to 8 ug / ml.

It is needless to say that it can be used by concentration under the condition of 0.5 to 128 ug / ml.

There is provided a composition for antimicrobial use of MRSA and / or VRSA containing a mixture of cryptotansinone and ampicillin as an active ingredient.

Here, the concentration of ampicillin is treated to be at least 8-1024 ug / ml, which is based on the MIC value for the MRSA or VRSA antimicrobial of oxacillin.

(2) a composition for antimicrobial use of MRSA and / or VRSA containing a mixture of cryptotansinone and oxacillin as an active ingredient.

Here, the concentration of oxacillin is treated to be at least 4 to 512 ug / ml, which is based on the MIC value for oxacillin MRSA or VRSA antimicrobial.

Depending on the conditions, the concentration of oxacillin can be adjusted to be at least 1 to 128 ug / ml, which is achieved by mixing the cryptotansinone 1/2 MIC value with vancomycin 1/2 MIC value for 3 to 6 hours Is based on exerting a synergistic effect such as completely inhibiting the bacteria.

(3) a composition for antimicrobial use of MRSA and / or VRSA containing a mixture of cryptotansinone and vancomycin as an active ingredient.

Here, the concentration of vancomycin is treated to correspond to at least 0.5 to 32 ug / ml, which is based on the MIC value for MRSA and / or VRSA of vancomycin.

Depending on the condition, the concentration of vancomycin can be adjusted to at least 0.125 to 8 ug / ml, which is achieved by mixing the cryptotansinone 1/2 MIC and vancomycin 1/2 MIC values after 3 to 6 hours It is based on exerting a synergistic effect such as completely inhibiting bacteria.

Example  3. MRSA  And / or VRSA Antimicrobial

(1) A pharmaceutical preparation is provided by using cryptoxanthin as an active ingredient and adding a pharmaceutically acceptable additive.

Here, the concentration of cryptoxanthin is used to be at least 0.5 to 64 ug / ml, which is based on the 1/2 MIC, MIC and MBC values for the antimicrobial activity of MRSA and / or VRSA of cryptoxanthin .

(2) A pharmaceutical preparation is provided by using a mixture of cryptotansinone and a conventional antimicrobial substance as an active ingredient and adding a pharmaceutically acceptable additive.

Here, the concentration of cryptoxanthin corresponds to at least 0.5 to 128 ug / ml, and the existing antimicrobial substance may be selected from all antibiotics. Preferably, at least one of ampicillin, oxacillin and vancomycin can be selected, but is not limited thereto.

In order to use the present invention as a pharmaceutical composition, it may be prepared by a known method in the pharmaceutical field, and may be mixed with the carrier itself, a pharmaceutically acceptable carrier, excipient, diluent or the like to prepare a powder, granule, Or injections, and the like. They may also be administered orally or parenterally.

The effective dose of the composition according to the present invention can be appropriately selected depending on the degree of absorption of the active ingredient in the body, the rate of water activation and excretion, the age, sex and condition of the patient, severity of the disease to be treated,

The composition according to the present invention can be used to prepare formulations such as a ring, a granule, a beverage, a tablet, a capsule, etc. In this case, it is a matter of course that a treating agent may be added to prepare each formulation.

Experimental Example  1. Minimum inhibitory concentration ( MIC ) Analysis experiment

1-1. Preparation for experiment

Bacteria used in this experiment were MSSA 25923, MRSA 33591, MSSA (Methicillin-sensitive Staphylococcus aureus 1, and MSSA 2, and the bacteria used as experimental groups are VRSA 3A063, VRSA 3A066, and MRSA 1 to 16.

Here, MSSA 25923 and MRSA 33591 are standard It is a reference strain.

The preparation of the experiment is as follows.

MSSA 25923, MRSA 33591, MSSA 1, MSSA 2, VRSA 3A063 and VRSA 3A066 were added to BHI (Brain-Heart Infusion) medium treated with 1% yeast extract (Difco Laboratories, Detroit, MRSA 1 to 16 were inoculated.

1-2. Experimental course

Liquid dilution test (broth dilution test) is a test to determine the activity of a specific antimicrobial agent against isolated bacteria. Liquid dilution culture methods include MIC (Minimum inhibitory concentration) and MBC (Minimun bactericidal contentration) measurements. The MIC is the minimum amount of antimicrobial agent needed to inhibit, or bacterium, bacterial growth.

MIC was repeated three times to measure the antimicrobial activity of cryptoxanthin and ampicillin. The MIC analysis method is as follows.

(1) Cryptotansinone, Ampicilin, Oxacilin, and Vancomycin were treated at the concentrations of the bacteria cultured by the procedure of Experimental Example 1-1.

Here, oxacillin is expected to have the same effect as methicillin as a component similar to methicillin, and is more stable than methicillin.

(2) The liquid medium was dispensed into a test tube, and the strain was cultured in a tube.

③ The tube was incubated at 37 ℃ for 18 ~ 48 hours, and the tube was observed with the naked eye to determine the lowest concentration of the tube that the growth of the strain was completely inhibited by MIC. MIC ₅₀ refers to a 50% minimum inhibitory concentration of bacterial growth, and MIC ₉₀ refers to a 90% minimum inhibitory concentration of bacterial growth.

1-3. Experiment result

Table 1 shows the results of MIC of the cryptoxanthin-treated group, the ampicillin treated group, the oxacillin treated group and the vancomycin treated group.

Bacterial species Cryptotansinone
(ug / mL) Ampicillin Oxacillin Vancomycin MIC ₅₀ < MIC ₉₀ < MIC MIC (ug / mL) MSSA ATCC 25923 4 64 64 8 0.25 0.5 MRSA ATCC 33591 0.5 4 4 512 8 One VRSA 3A063 0.5 2 2 512 16 VRSA 3A066 One 4 4 128 512 32 MSSA 1 2 16 16 1024 0.5 One MSSA 2 8 32 32 256 One One MRSA 1 2 16 16 128 128 One MRSA 2 8 64 64 256 8 2 MRSA 3 One 4 4 128 128 2 MRSA 4 0.5 4 4 128 512 One MRSA 5 2 64 64 128 512 One MRSA 6 0.5 4 4 64 64 One MRSA 7 2 8 8 128 512 One MRSA 8 4 32 32 256 512 One MRSA 9 2 8 8 64 512 One MRSA 10 2 16 16 128 512 One MRSA 11 4 8 8 64 64 One MRSA 12 One 4 4 128 128 One MRSA 13 4 32 32 128 32 One MRSA 14 0.5 8 8 64 8 One MRSA 15 0.5 4 4 64 128 One MRSA 16 2 8 8 128 128 One

The results of the measurement showed that the MICs when treated with cryptoxanthin, ampicillin and oxacillin or vancomycin in the experimental groups (VRSA 3A063, VRSA 3A066 and MRSA 1 to 16) were compared with the control group (MSSA ATCC 25923, MRSA ATCC 33591, MSSA 1, MSSA 2) was generally lower than the MIC when treated with cryptoxanthin, ampicillin and oxacillin or vancomycin.

The MIC of the cryptoxanthin-treated group was at least 2 ug / ml to a maximum of 64 ug / ml. The MIC values of the cryptoxanthin-treated group and the ampicillin-treated group were significantly lower than those of the cryptoxanthin treated group when the ampicillin treated group had a minimum of 8 ug / ml and a maximum of 1024 ug / ml, Able to know.

In addition, the MIC of oxacillin ranged from a minimum of 0.25 μg / ml to a maximum of 512 μg / ml, and the MIC of vancomycin ranged from a minimum of 0.5 μg / ml to a maximum of 32 μg / ml.

The lower the MIC value, the higher the susceptibility to antibiotics. The higher susceptibility to antibiotics means higher antimicrobial activity. Thus, it can be seen that cryptotansinone has a higher antimicrobial activity than ampicillin, and the antimicrobial activity is similar to that of oxacillin.

Given that cryptotanshinone is a natural product, the fact that cryptoxanthin has a similar or higher degree of MRSA or VRSA antimicrobial activity to synthetic drugs suggests that the antimicrobial activity of cryptoxanthin is very high.

Experimental Example  2. Minimum sterilization concentration ( MBC ) Analysis experiment

2-1. Preparation for experiment

The same procedure as in Experimental Example 1-1 was carried out.

2-2. Experimental course

As described in Experimental Example 1-2, the liquid dilution test is a test to determine the activity of a specific antimicrobial agent against the separated bacteria. The liquid dilution culture method has a minimum inhibitory concentration (MIC) There is an MBC (Minimun bactericidal contentration) measurement.

MBC is the minimum amount of antimicrobial agent required to kill bacteria and is defined as the minimum concentration of antibiotics that reduces the amount of pathogens by 99.9% (ie, 10 ^{5 to 6} / mL to less than 10 ^{2 to 3} / mL). Most of the antimicrobial antimicrobials decrease the bacteria by more than 99.9% at the concentration of MIC, so the majority of MIC and MBC are the same.

In general, it is reasonable to predict the sterilization by MIC alone. However, if it is necessary to use antibiotics with high sterilization power to remove the causative organism due to defects before host defense such as meningitis, endocarditis, neutropenia, MBC It is used as an indicator.

MBC was performed to measure the antimicrobial activity of cryptotansenoside. The MBC analysis method is as follows.

(1) For the MIC analysis, the medium cultured in the procedure of Experimental Example 1-2 was visually observed, and the medium in which the growth of the strain was completely inhibited was selected and spread on a 96-well plate.

② The number of live bacteria was measured in the medium.

2-3. Experiment result

Table 2 shows the MBC values of the cryptoxanthin-treated group, the ampicillin treated group, the oxacillin treated group, and the vancomycin treated group.

Bacterial species Cryptotansinone
(ug / mL) Ampicillin Oxacillin Vancomycin MBC MBC (ug / mL) MSSA ATCC 25923 256 16 One 2 MRSA ATCC 33591 16 2048 16 4 VRSA 3A063 4 2048 1024 32 VRSA 3A066 16 256 1024 64 MSSA 1 64 2048 One 4 MSSA 2 128 512 2 2 MRSA 1 32 256 256 2 MRSA 2 128 256 32 4 MRSA 3 16 512 512 4 MRSA 4 8 256 2048 2 MRSA 5 128 512 1024 2 MRSA 6 16 128 256 2 MRSA 7 16 256 1024 4 MRSA 8 64 256 2048 4 MRSA 9 16 128 1024 2 MRSA 10 64 256 1024 2 MRSA 11 32 128 128 2 MRSA 12 8 256 256 4 MRSA 13 128 256 128 2 MRSA 14 16 128 16 4 MRSA 15 16 128 256 4 MRSA 16 16 512 512 2

As a result of the measurement, as in the MIC measurement results of Experimental Example 1, MBCs when treated with cryptoxanthin, ampicillin and oxacillin and vancomycin in the experimental groups (MRSA 1 to 16) were compared with the control group (MSSA ATCC 25923 , MRSA ATCC 33591, VRSA 3A063, VRSA 3A066, MSSA 1 and MSSA 2) were significantly lower than MBC when treated with cryptotansinone, ampicillin, oxacillin and vancomycin.

MBC values of cryptoxanthin-treated group were at least 4 ug / ml and maximal 256 ug / ml. The MBC values of the cryptoxanthin-treated group and the ampicillin-treated group were lower than those of the cryptoxanthin-treated group and the ampicillin-treated group, respectively, from a minimum of 16 ug / ml to a maximum of 2048 ug / ml.

The MBC value of oxacillin was 2048 ug / ml at a minimum of 1 ug / ml, and the MBC value of vancomycin was at least 2 ug / ml to a maximum of 64 ug / ml.

Like the MIC, the lower the MBC value, the higher the susceptibility to antibiotics, ie, the higher the antibacterial activity. Thus, it can be seen that cryptotansinone has a higher antimicrobial activity than ampicillin, and the antimicrobial activity is similar to that of oxacillin.

Given that cryptotanshinone is a natural product, the fact that cryptoxanthin has a similar or higher degree of MRSA or VRSA antimicrobial activity to synthetic drugs suggests that the antimicrobial activity of cryptoxanthin is very high.

Experimental Example  3. checkers  Board dilution ( Checker - Board dilution ) Experiment

3-1. Preparation for experiment

The same procedure as in Experimental Example 1-1 was carried out.

3-2. Experimental course

Synergistic action is a phenomenon in which the drug efficacy when two different drugs are administered simultaneously in a certain dose is stronger than the sum of the drug efficacy when each drug is administered alone and the additive ) Action is the same or similar phenomenon in which the sum of the drug efficacy when two different drugs are simultaneously administered in a certain dose and the drug efficacy when each drug is administered alone is the same or similar.

Fractional inhibitory concentration index (FICI) is the sum of the FIC (Fractional inhibitory concentration) values of the most effective combinations and is most commonly used to interpret the results of antibiotic combination studies. When the FICI is less than 0.5, the synergistic effect is obtained. When the FICI is 2.0 or more, the antagonistic action is judged.

The following equation (1) is a calculation method of FIC.

Here, (A) refers to the MIC when the drug to be used in combination with the drug A is used in combination, and MIC _A means the MIC when the drug A is administered alone.

Equation (2) below is a calculation method of FICI.

FBCI (fractional bactericidal concentration index) is the sum of the values of FBC (fractional bactericidal concentration) in the most effective combination. The calculation method is the same as the calculation method of FICI.

Experiments in which the synergistic effect was measured by mixing ampicillin, oxacillin or vancomycin in cryptoxanthin was performed with Checker-Board Dilution.

(1) The same procedure as (1) in Experimental Example 1-1 was carried out.

(2) Each mixture was prepared by treating cryptotanshinone with ampicillin, oxacillin, and vancomycin in combination.

③ The prepared mixture was serially diluted.

(4) Treat 96 well microplates with Mueller-Hinton liquid medium supplemented with cations and then treat the series diluted mixture.

⑤ Sequence The diluted mixture is treated with the culture solution cultured on the treated medium.

⑥ The medium treated with the strain was incubated at 37 ° C for 24 hours.

⑦ The MIC and MBC analyzes were performed in the same manner as in Experimental Examples 1 and 2.

3-3. Experiment result

Table 3 shows the results of a checkerboard dilution experiment when the cryptotansinone was treated alone and when the mixture of cryptoxanthin and ampicillin was treated.

Bacterial species
Treated drug MIC / MBC (ug / ml)
FIC / FBC
FICI / FBCI
result Exclusive mix MSSA ATCC
25923 Cryptotansinone 64/256 8/32 0.125 / 0.125 0.375 / 0.375
Synergistic
/ Synergistic
Oxacillin 0.25 / 1 0.0625 / 0.25 0.25 / 0.25 MRSA ATCC
33591
Cryptotansinone
4/16
1/4 0.25 / 0.25
0.5 / 0.5
Synergistic
/ Synergistic
Oxacillin 8/16 2/4 0.25 / 0.25 VRSA 3A063 Cryptotansinone
2/4
0.5 / 2 0.25 / 0.5
0.375 / 0.375
Synergistic
/ Synergistic
Oxacillin 512/1024 64/128 0.125 / 0.125 VRSA 3A066
Cryptotansinone 4/16 1/4 0.25 / 0.25 0.375 / 0.5 Synergistic
/ Synergistic Oxacillin 512/1024 64/256 0.125 / 0.25 MSSA 1 Cryptotansinone 16/64 2/8 0.125 / 0.125 0.375 / 0.25 Synergistic
/ Synergistic Oxacillin 0.5 / 1 0.125 / 0.125 0.25 / 0.125 MSSA 2 Cryptotansinone 32/128 16/32 0.5 / 0.25 0.375 / 0.375 Synergistic
/ Synergistic Oxacillin 1/2 0.125 / 0.25 0.125 / 0.125 MRSA 1
Cryptotansinone 16/32 4/16 0.25 / 0.5 0.5 / 0.75
Synergistic
/ Appropriately Oxacillin 128/256 32/64 0.25 / 0.25 MRSA 2
Cryptotansinone 64/128 16/16 0.5 / 0.125 0.5 / 0.375 Synergistic
/ Synergistic Oxacillin 8/32 2/8 0.25 / 0.25 MRSA 3 Cryptotansinone 4/16 0.5 / 4 0.125 / 0.25 0.375 / 0.375 Synergistic
/ Synergistic Oxacillin 128/512 32/64 0.25 / 0.125 MRSA 4 Cryptotansinone 4/8 2/2 0.5 / 0.25 0.75 / 0.375
Appositional
/ Synergistic Oxacillin 512/2048 128/256 0.25 / 0.125 MRSA 5 Cryptotansinone 64/128 16/32 0.25 / 0.25 0.375 / 0.5
Synergistic
/ Synergistic Oxacillin 512/1024 64/256 0.125 / 0.25 MRSA 6
Cryptotansinone 4/16 1/4 0.25 / 0.25 0.5 / 0.5
Synergistic
/ Synergistic Oxacillin 64/256 16/64 0.25 / 0.25 MRSA 7 Cryptotansinone 8/16 2/8 0.25 / 0.5 0.375 / 0.75 Synergistic
/ Appropriately Oxacillin 512/1024 64/256 0.125 / 0.25 MRSA 8 Cryptotansinone 32/64 8/32 0.25 / 0.5 0.5 / 0.625 Synergistic
/ Synergistic Oxacillin 512/2048 128/256 0.25 / 0.125 MRSA 9
Cryptotansinone 8/16 2/4 0.375 / 0.3125
Synergistic
/ Synergistic Oxacillin 512/1024 64/64 0.25 / 0.25 MRSA 10 Cryptotansinone 16/64 2/8 0.125 / 0.0625 0.3125 / 0.25 Synergistic
/ Synergistic Oxacillin 512/1024 32/128 0.125 / 0.125 MRSA 11 Cryptotansinone 8/32 1/4 0.0625 / 0.125 0.375 / 0.375 Synergistic
/ Synergistic Oxacillin 64/128 16/64 0.125 / 0.125 MRSA 12
Cryptotansinone 4/8 1/2 0.25 / 0.25 0.5 / 0.5 Synergistic
/ Synergistic Oxacillin 128/256 32/64 0.25 / 0.25 MRSA 13
Cryptotansinone 32/128 8/32 0.25 / 0.25 0.5 / 0.375
Synergistic
/ Synergistic Oxacillin 32/128 8/8 0.25 / 0.125 MRSA 14
Cryptotansinone 8/16 2/4 0.25 / 0.25 0.375 / 0.5 Synergistic
/ Synergistic Oxacillin 8/16 1/4 0.125 / 0.25 MRSA 15
Cryptotansinone 4/16 1/8 0.25 / 0.5 0.5 / 0.75 Synergistic
/ Appropriately Oxacillin 128/256 32/64 0.25 / 0.25 MRSA 16 Cryptotansinone 8/16 2/4 0.25 / 0.25 0.75 / 0.375 Moody Oxacillin 128/512 64/64 0.5 / 0.125

As a result, the cryptoxanthinone-treated group had a MIC of at least 2 ug / ml to a maximum of 64 ug / ml when treated with cryptoxanthinone alone and a MBC of at least 4 ug / ml to a maximum of 128 ug / ml Respectively.

The MIC of the cryptoxanthinone-comparative group ranged from a minimum of 0.25 μg / ml to a maximum of 8 μg / ml and the cryptoxanthin-comparative group had a minimum of 1 μg / ml to a maximum of 16 μg / ml.

The MIC of the oxacillin-treated group when treated with oxacillin alone was at least 4 ug / ml up to 512 ug / ml and the MBC was at least 16 ug / ml up to 2048 ug / ml.

The MIC of the oxacillin-comparative group was at least 4 ug / ml up to 64 ug / ml and the MBC was at least 16 ug / ml up to 256 ug / ml.

The MIC of the cryptoxanthinone-treated group when combined with cryptotansinone and oxacillin was at least 0.5 ug / ml up to 16 ug / ml and the MBC was at least 1 ug / ml up to 16 ug / ml.

The MIC of the cryptoxanthinone-comparative group was at least 1 ug / ml up to 16 ug / ml, and the MBC was at least 4 ug / ml up to 32 ug / ml.

The MIC of the oxacillin-treated group when combined with cryptoxanthin and oxacillin was at least 128 ug / ml at a minimum of 1 ug / ml and the maximum of 256 ug / ml at a minimum of 4 ug / ml.

The MIC of the oxacillin-comparator group ranged from 0.0625 ug / ml to a maximum of 2 ug / ml and the MBC was at least 0.125 ug / ml to a maximum of 4 ug / ml.

The cryptotansinone-treated group had a FIC of at least 0.25 ug / ml up to 0.5 ug / ml and the FBC was also at least 0.25 ug / ml up to 0.5 ug / ml.

The cryptotansinone-comparative group had a FIC of at least 0.25 ug / ml up to 0.5 ug / ml and a FBC of at least 0.25 ug / ml up to 0.5 ug / ml.

The oxacillin-treated group had a minimum FIC of 0.0625 ug / ml up to 0.5 ug / ml, and the FBC also had a minimum of 0.0625 ug / ml up to 0.5 ug / ml.

The FIC of the oxacillin-comparative group was at least 0.125 ug / ml up to 0.25 ug / ml and the FBC was also at least 0.125 ug / ml up to 0.25 ug / ml.

The FICI of cryptoxanthin and oxacillin - treated groups ranged from at least 0.3125 ug / ml up to 0.5 ug / ml, and 10 of the 12 groups showed a synergistic effect. FICI when mixed with cryptotansinone and oxacillin in the experimental group is generally synergistic.

The FBCI of cryptoxanthin and oxacillin - treated groups was at least 0.25 ug / ml to 0.5 ug / ml at maximum, and 10 out of 12 groups showed synergistic effect. In the experimental group, FBCI when mixed with cryptotansinone and oxacillin is generally highly synergistic.

In the cryptoxanthin and oxacillin - comparative groups, the FICI ranged from at least 0.375 ug / ml up to 0.5 ug / ml, and all four groups showed a synergistic effect. When the cryptotansinone and oxacillin were mixed in the comparative group, the FICI was highly synergistic.

The FBCI of the cryptoxanthin and oxacillin - comparative groups were at least 0.25 ug / ml to 0.5 ug / ml at maximum, and all four groups showed a synergistic effect. When the cryptotansinone and oxacillin were mixed in the comparative group, the synergistic effect of FBCI was high.

MIC, MBC, and FIC, and FBC, FIC, and FBCI when cryptotanshinone and oxacillin were treated alone were compared with those of MIC, MBC, FIC and FBC, FICI, FICI and FBCI values are lower and mixing of cryptotansinone and oxacillin shows that bacterial growth inhibition and bactericidal effects are further increased.

The MIC, MBC, FIC, FBC, FICI and FBCI values of the experimental group and the comparative group were similar to each other or lower in the experimental group.

The comparative group showed high susceptibility to antibiotics when treated with any antibiotics, suggesting that the experimental group had a similar or low value to the comparative group and that the cryptotansinone was highly effective when used alone or in combination.

Table 4 shows the results of a checkerboard test when cryptotanshinone alone was treated and when a mixture of cryptoxanthinone and vancomycin was treated.

Bacterial species Treated drug MIC / MBC FIC / FBC FICI / FBCI outcome Exclusive mix MSSA ATCC 25923 Cryptotansinone 64/256 16/64 0.25 / 0.25 0.5 / 0.5 Synergistic / Ampicillin 0.5 / 2 0.125 / 0.5 0.25 / 0.25 Synergistic MRSA ATCC 33591 Cryptotansinone 4/16 1/4 0.25 / 0.25 0.5 / 0.5 Synergistic / Ampicillin 1/4 0.25 / 1 0.25 / 0.25 Synergistic VRSA 3A063 Cryptotansinone 2/4 0.5 / 1 0.125 / 0.25 0.375 / 0.5
Synergistic / Ampicillin 16/32 4/8 0.25 / 0.25 Synergistic VRSA 3A066 Cryptotansinone 4/16 1/4 0.25 / 0.25 0.5 / 0.5
Synergistic / Ampicillin 32/64 8/16 0.25 / 0.25 Synergistic MSSA 1
Cryptotansinone 16/64 4/16 0.25 / 0.25 0.5 / 0.75
Synergistic / Ampicillin 1/4 0.25 / 2 0.25 / 0.5 Appositional MSSA 2
Cryptotansinone 32/128 8/16 0.25 / 0.125 0.5 / 0.625
Synergistic / Ampicillin 1/2 0.25 / 1 0.25 / 0.5 Appositional MRSA 1
Cryptotansinone 16/32 4/8 0.25 / 0.25 0.5 / 0.5
Synergistic / Ampicillin 1/2 0.25 / 0.5 0.25 / 0.25 Synergistic MRSA 2
Cryptotansinone 64/128 16/32 0.25 / 0.25 0.5 / 0.75
Synergistic / Ampicillin 2/4 0.5 / 2 0.25 / 0.5 Appositional MRSA 3
Cryptotansinone 4/16 1/4 0.25 / 0.25 0.375 / 0.5
Synergistic / Ampicillin 2/4 0.25 / 1 0.125 / 0.25 Synergistic MRSA 4
Cryptotansinone 4/8 1/2 0.25 / 0.25 0.5 / 0.5
Synergistic / Ampicillin 1/2 0.25 / 0.5 0.25 / 0.25 Synergistic / MRSA 5
Cryptotansinone 64/128 16/32 0.25 / 0.25 0.5 / 0.5
Synergistic / Ampicillin 1/2 0.25 / 0.5 0.25 / 0.25 Synergistic MRSA 6
Cryptotansinone 4/16 2/4 0.5 / 0.25 0.75 / 0.5
In addition, Ampicillin 1/2 0.25 / 0.5 0.25 / 0.25 Synergistic MRSA 7
Cryptotansinone 8/16 8/16 0.25 / 0.25 0.5 / 0.375
Synergistic / Ampicillin 1/4 0.25 / 0.5 0.25 / 0.125 Synergistic MRSA 8
Cryptotansinone 32/64 2/4 0.25 / 0.25 0.5 / 0.375
Synergistic / Ampicillin 1/4 0.25 / 0.5 0.25 / 0.125 Synergistic MRSA 9
Cryptotansinone 8/16 8/16 0.25 / 0.25 0.5 / 0.5
Synergistic / Ampicillin 1/2 0.25 / 0.5 0.25 / 0.25 Synergistic MRSA 10
Cryptotansinone 16/64 4/8 0.25 / 0.25 0.5 / 0.75
Synergistic / Ampicillin 1/2 0.25 / 1 0.25 / 0.5 Appositional MRSA 11
Cryptotansinone 8/32 2/8 0.25 / 0.25 0.75 / 0.5
In addition, Ampicillin 1/2 0.5 / 0.5 0.5 / 0.25 Synergistic MRSA 12
Cryptotansinone 4/8 1/2 0.25 / 0.25 0.5 / 0.375
Synergistic / Ampicillin 1/4 0.25 / 0.5 0.25 / 0.125 Synergistic MRSA 13
Cryptotansinone 32/128 16/32 0.25 / 0.25 0.5 / 0.5
Synergistic / Ampicillin 1/2 0.25 / 0.5 0.25 / 0.25 Synergistic MRSA 14
Cryptotansinone 8/16 2/4 0.25 / 0.25 0.5 / 0.5
Synergistic / Ampicillin 1/4 0.25 / 1 0.25 / 0.25 Synergistic MRSA 15
Cryptotansinone 4/16 2/4 0.5 / 0.125 0.75 / 0.375
In addition, Ampicillin 1/4 0.25 / 0.5 0.25 / 0.125 Synergistic MRSA 16
Cryptotansinone 8/16 2/4 0.25 / 0.25 0.375 / 0.5
Synergistic / Ampicillin 1/2 0.125 / 0.5 0.125 / 0.25 Synergistic

As a result, the cryptoxanthinone-treated group had a MIC of at least 2 ug / ml to a maximum of 64 ug / ml when treated with cryptoxanthinone alone and a MBC of at least 4 ug / ml to a maximum of 128 ug / ml Respectively.

The MIC of the cryptoxanthinone-comparative group ranged from a minimum of 0.25 μg / ml to a maximum of 8 μg / ml and the cryptoxanthin-comparative group had a minimum of 1 μg / ml to a maximum of 16 μg / ml.

The MIC of the ampicillin-treated group when treated with ampicillin alone was at least 32 ug / ml up to 512 ug / ml and the MBC was at least 64 ug / ml up to 2048 ug / ml.

The MIC of the ampicillin-comparator group ranged from at least 8 μg / ml up to 512 μg / ml and the MBC was at least 16 μg / ml up to 2048 μg / ml.

The cryptoxanthinone-treated group had a MIC of at least 0.5 ug / ml to a maximum of 16 ug / ml, and a MBC of at least 2 ug / ml to a maximum of 32 ug / ml when cryptoxanthin and ampicillin were used in combination.

The MIC of the cryptoxanthinone-comparative group was at least 1 ug / ml up to 16 ug / ml, and the MBC was at least 8 ug / ml up to 64 ug / ml.

The MIC of the ampicillin-treated group when cryptoxanthin and ampicillin were combined was at least 8 ug / ml up to 128 ug / ml and the MBC was at least 8 ug / ml up to 256 ug / ml.

The MIC of the ampicillin-comparative group was at least 2 ug / ml up to 128 ug / ml and the MBC was at least 8 ug / ml up to 512 ug / ml.

The cryptotansinone-treated group had a FIC of at least 0.125 ug / ml up to 0.5 ug / ml and the FBC was also at least 0.125 ug / ml up to 0.5 ug / ml.

The FIC of the cryptoxanthinone-comparative group was 0.25 ug / ml, and the FBC was 0.125 ug / ml to 0.25 ug / ml.

The FIC of the ampicillin-treated group was at least 0.125 ug / ml up to 0.5 ug / ml and the FBC was also at least 0.125 ug / ml up to 0.5 ug / ml.

The FIC of the ampicillin-comparator group was 0.25 ug / ml, and the FBC was 0.25 ug / ml to 0.5 ug / ml.

The FICI of cryptoxanthin and ampicillin - test groups ranged from at least 0.375 ug / ml to a maximum of 0.75 ug / ml, with a synergistic effect in 11 out of 14 groups. FICI when mixed with cryptotansinone and oxacillin in the experimental group is generally synergistic.

The FBCI of cryptoxanthin and ampicillin - treated groups ranged from 0.25 ug / ml to 0.75 ug / ml at the maximum, and 11 of 14 groups showed a synergistic effect. In the experimental group, FBCI when mixed with cryptotansinone and oxacillin is generally highly synergistic.

The FICI of the cryptoxanthin and ampicillin - comparative groups was 0.5, and all four groups showed a synergistic effect. When the cryptotansinone and oxacillin were mixed in the comparative group, the FICI was highly synergistic.

The FBCI of the cryptoxanthin and oxacillin - comparative groups ranged from a minimum of 0.375 ug / ml to a maximum of 0.75 ug / ml, with a synergistic effect in three of the four groups. The FBCI when mixed with cryptotansinone and oxacillin in the comparative group is generally highly synergistic.

MIC, MBC, FIC, FBC, FICI and FBCI when treated with a mixture of cryptotansinone and ampicillin, compared with the values of MIC, MBC, FIC and FBC, FICI and FBCI when treated with cryptoxanthin and ampicillin alone FBCI values are lower, and the combination of cryptoxanthin and ampicillin indicates that bacterial growth inhibition and bactericidal effects are further increased.

The MIC, MBC, FIC, FBC, FICI and FBCI values of the experimental group and the comparative group were similar to each other or lower in the experimental group.

As mentioned above, the comparative group showed high antimicrobial susceptibility even after treatment with any antibiotics. In the experimental group, similar or lower values than the comparative group showed that the cryptotansinone was highly effective when used alone or in combination I will suggest.

Experimental Example  4. Time kill ( Time kill ) Curve experiment

4-1. Preparation for experiment

The same procedure as in Experimental Example 1-1 was carried out.

4-2. Experimental course

Time-kill curves for oral bacteria were run with a broth macrodilution experiment. The experimental method is as follows.

1) BHI medium was cultured by setting the bacterial counts (MSSA ATCC 25923, MRSA ATCC 33591, VRSA 3A063, VRSA 3A066, MSSA 1 and MSSA 2) and experimental groups (MRSA 1 to 16) to 1 × 10 ⁵ CFU / ml .

② BHI medium was divided into 4 groups, and MIC cryptotansinone, 1/2 MIC cryptoxanthinone + 1/2 MIC oxacillin, 1/2 MIC cryptoxanthinone + 1/2 MIC ampicillin, 1/2 MIC Treated with cryptoxanthinone + 1/2 MIC vancomycin.

The medium was incubated at 37 ° C. And then at 0, 30, 1, 2, 3, 4, 5, 6, and 12 hours after incubation.

(4) The number of colonies of each medium was calculated twice to obtain the average value of the colonies.

4-3. Experiment result

Figure 1 shows the results of treatment of cryptotansinone, cryptoxanthin and ampicillin, cryptotansonone and oxacillin, cryptoxanone and vancomycin in MSSA 25923, MRSA 33591, MSSA 1 and MSSA 2, and VRSA 1 and VRSA 2 FIG. 4 is a graph showing the antimicrobial effect of the composition according to the present invention. FIG.

FIG. 2 shows the results of treatment of cryptotansinone, cryptoxanthin and ampicillin, cryptotansonone and oxacillin, cryptoxanthone and vancomycin in MRSA 1, MRSA 2, MRSA 3, MRSA 4, MRSA 5 and MRSA 6 FIG. 4 is a graph showing the antimicrobial effect of the composition according to the present invention. FIG.

Figure 3 shows the results of treatment of cryptotansinone, cryptotansinone and ampicillin, cryptotansinone and oxacillin, cryptoxanthone and vancomycin in MRSA 7, MRSA 8, MRSA 9, MRSA 10, MRSA 11 and MRSA 12, FIG. 4 is a graph showing the antimicrobial effect of the composition according to the present invention. FIG.

Figure 4 shows the antimicrobial effect patterns of MRSA 13, MRSA 14, MRSA 15 and MRSA 16 after treatment with cryptotansinone, cryptoxanthin and ampicillin, cryptotansinone and oxacillin, cryptoxanthinone and vancomycin, respectively Fig.

The results showed that the highest antimicrobial activity of MSSA 25923 was the combination of cryptoxanthin and ampicillin, followed by cryptoxanthin and vancomycin, cryptoxanthin and oxacillin, The combined treatment group and the cryptosanthinone treated group were higher in order of antibacterial activity.

The time-kill curves of MRSA 33591 showed the highest antimicrobial activity in cryptoxanthin and ampicillin treated groups, followed by cryptotansonone and oxacillin combined treatment, cryptoxanthin and vancomycin , And cryptoxanthinone treated groups were higher in order of antibacterial activity.

The time-kill curves of MSSA 1 showed the highest antimicrobial activity in combination with cryptoxanthin and oxacillin, followed by cryptotansinone plus ampicillin, cryptotansinone and vancomycin, The antimicrobial activity was higher in order of tanshinone treatment group.

The time-kill curves of MSSA 2 showed the highest antimicrobial activity in combination with cryptotansinone and vancomycin, followed by cryptotansonone plus oxacillin, cryptotansonone plus ampicillin Treated group, and cryptoxanthinone treated group, respectively.

The time-kill curves of VRSA 1 showed the highest antimicrobial activity in combination with cryptotansinone and vancomycin, followed by cryptotansinone plus oxacillin, cryptotansonone plus ampicillin Treated group, and cryptoxanthinone treated group.

The time-kill curves of VRSA 2 showed the highest antimicrobial activity in combination with cryptoxanthin and vancomycin, followed by combination of cryptotansonone and oxacillin, combination of cryptoxanthin and ampicillin Treated group, and cryptoxanthinone treated group, respectively.

The time-kill curves of MRSA 1 showed the highest antimicrobial activity in combination with cryptoxanthin and oxacillin, followed by cryptotansinone plus vancomycin, cryptanthinone plus ampicillin Treated group, and cryptoxanthinone treated group, respectively.

The time-kill curves of MRSA 2 showed the highest antimicrobial activity in combination with cryptoxanthin and oxacillin, followed by cryptotansonone plus vancomycin, cryptanthinone plus ampicillin Treated group, and cryptoxanthinone treated group, respectively.

The time-kill curves of MRSA 3 showed the highest antimicrobial activity in combination with cryptoxanthin and ampicillin, followed by cryptotansonone and vancomycin, cryptoxanthin and oxacillin Treated group, and cryptoxanthinone treated group, respectively.

The time-kill curves of MRSA 4 showed the highest antimicrobial activity in combination with cryptoxanthin and oxacillin, followed by cryptotansinone plus ampicillin, cryptotansonone plus vancomycin Treated group, and cryptoxanthinone treated group, respectively.

When the time-kill curve of MRSA 5 was examined, the antibacterial activity of the combination of cryptotansinone and vancomycin, the combination of cryptotansinone and ampicillin, and the combination of cryptotansinone and oxacillin were similar, The antimicrobial activity of the non - treated group was the lowest.

The time-kill curves of MRSA 6 showed the highest antimicrobial activity in combination with cryptoxanthin and vancomycin, followed by cryptotansonone plus oxacillin, cryptoxanthin and ampicillin Treated group, and cryptoxanthinone treated group, respectively.

The time-kill curves of MRSA 7 showed the highest antimicrobial activity in combination with cryptotansinone and oxacillin, followed by cryptotansonone plus vancomycin, cryptotansonone plus ampicillin Treated group, and cryptoxanthinone treated group, respectively.

The time-kill curves of MRSA 8 showed the highest antimicrobial activity in combination with cryptoxanthin and vancomycin, followed by cryptotansonone plus oxacillin, cryptanthinone plus ampicillin Treated group, and cryptoxanthinone treated group, respectively.

When the time-kill curve of MRSA 9 was examined, the antibacterial activity of the combination of cryptotansinone and vancomycin, the combination of cryptoxanthin and ampicillin, and the combination of cryptotansinone and oxacillin were similar, The antimicrobial activity of the non - treated group was the lowest.

The time-kill curves of MRSA 10 showed the highest antimicrobial activity in combination with cryptoxanthin and oxacillin, followed by cryptotansinone plus vancomycin, cryptanthinone plus ampicillin Treated group, and cryptoxanthinone treated group, respectively.

The time-kill curve of MRSA 11 showed the highest antimicrobial activity in combination with cryptoxanthin and ampicillin, followed by cryptotansonone with vancomycin, cryptoxanthin with oxacillin Treated group, and cryptoxanthinone treated group, respectively.

The time-kill curves of MRSA 12 showed similar antibacterial activities against the combination of cryptotansinone and vancomycin, cryptotansinone and ampicillin, and cryptotansinone and oxacillin, The antimicrobial activity of the non - treated group was the lowest.

The time-kill curves of MRSA 13 showed the highest antimicrobial activity in combination with cryptoxanthin and ampicillin, followed by cryptoxanthin and vancomycin. The combination of cryptotansinone and oxacillin and the cryptoxanthin treatment group showed higher antimicrobial activity.

The time-kill curves of MRSA 14 showed the highest antimicrobial activity in combination with cryptoxanthin and ampicillin, followed by cryptotansonone plus vancomycin, cryptotansonone plus oxacillin Treated group, and cryptoxanthinone treated group, respectively.

The time - kill curves of MRSA 15 showed the highest antimicrobial activity in combination with cryptoxanthin and vancomycin. The antimicrobial activity of cryptoxanthin and oxacillin was similar to that of vancomycin. Next, the antibacterial activity was higher in the order of cryptotansinone and ampicillin combined treatment and cryptoxanthin treated group.

When the time-kill curve of MRSA 16 was examined, the antibacterial activity of the combination of cryptotansinone and vancomycin, the combination of cryptoxanthin and ampicillin, and the combination of cryptotansinone and oxacillin were similar, The antimicrobial activity of the non - treated group was the lowest.

In summary, it can be seen that cryptoxanthinone and existing antimicrobials have the effect of causing a synergistic effect on MRSA and VRSA.

In other words, the cryptotansinone 1/2 MIC value and the mixture of the existing antimicrobial substance are treated, and after 3 ~ 6 hours, the growth of MRSA and VRSA is completely weakened.

Claims (5)

delete A composition for antibiosis of VRSA (Vancomysin Resistant Staphylococcus Aureus) containing cryptotansinone as an active ingredient.
The method of claim 2,
A composition for the antibacterial activity of VRSA (Vancomysin Resistant Staphylococcus Aureus) comprising as an active ingredient a mixture of the above cryptotansinone and at least one antimicrobial selected from oxacillin, ampicillin or vancomycin.
The method of claim 2,
Wherein the concentration of cryptoxanthin is from 0.5 to 128 ug / ml.
A pharmaceutical preparation for the antibacterial activity of VRSA (Vancomysin Resistant Staphylococcus Aureus) to which a composition according to any one of claims 2 to 4 is added with a pharmaceutically acceptable additive.

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