KR20040105110A - Anti-microbial composition comprising pinosylvin and method for synthesizing the pinosylvin - Google Patents

Abstract

PURPOSE: Provided are an anti-microbial composition useful for living goods and food preservatives requiring both physiological and bactericidal action, and a process for synthesizing pinosylvin in large quantities. CONSTITUTION: The anti-microbial composition comprises pinosylvin of the formula I as an effective ingredient. The pinosylvin is synthesized from pine needles by the process comprising the steps of: subjecting a mixture of benzylbromide and triethyl phosphite to TLC and distillation to obtain diethylbenzyl phosphite; adding 3,5-dimethoxybenzyl aldehyde to a mixture of the obtained diethylbenzyl phosphite, dimethyl formamide and sodium methoxide which are cooled under nitrogen gas; subjecting the mixture to TLC and addition of a mixture of water and methanol to precipitate the reaction product; purifying a filtrate by chromatography to obtain 3,5-dimethoxystilbene; extracting a mixture of 3,5-dimethoxystilbene and pyridine hydrochloride using HCl and ether solution; removing water and solvent from ether layer; and purifying the product by column chromatography.

Description

Anti-microbial composition comprising pinosylvin and method for synthesizing the pinosylvin}

The present invention relates to a composition for an antimicrobial comprising a stilbene compound of the pine needle extract component, a pinocylbin as an active ingredient, and a method for synthesizing the pinocylbin. More specifically, the present invention synthesizes a large amount of stilbene-based compound known as phytoalexin (phytoalexin) in a large amount by the following process, microorganisms and fungi present in the air of the synthesized stilbene-based compound, and bacteria causing food poisoning In addition, the present invention relates to the development of the compound as a household goods and food preservatives by verifying the growth inhibitory or bactericidal efficacy against fungi and the like, which are infected by fruits.

Stilbene-based compounds present in pine trees were originally known as phytoalexin, a defense substance that inhibits the growth of bacteria and fungi that invaded from the outside in plants. Stilbene compounds are produced only in a few plants, so far known as Polygonum cuspidatum , Eucalyptus, Scottish pine, spruce, grapes, Arachis hypogaea , lilies ( lily). In these plants, stilbene compounds are synthesized from malony-CoA and p-coumaroyl-CoA or p-cinnamoyl-CoA by stilbene synthase, and their synthesis from external environmental stresses such as mold, UV and ozone. Induction (Schoeppner and Kindl H, 1979; Fliegmann et al., 1992; Fritzemeier et al., 1983; Vornam et al., 1988). In recent years, multifaceted studies on the biosynthetic mechanism and regulation of this substance have been conducted at the molecular and genetic level. As a result, genes involved in biosynthesis of this substance were isolated from pine, grape, and peanut (Sparvoli et al., 1994; Preisig-Muller et al., 1999; Schubert et al., 1997). Much information has been accumulated.

On the other hand, studies on stilbenes similar to those of pine in grapes have been around for a long time, and the antistress action of these compounds is well known (Langcake et al., 1979). In addition, there have been many cases of developing and reporting disease-resistant transformant crops such as antifungal, antibacterial, antiviral, and antiworm by introducing synthetic genes using the stilbene's antistress action and stress resistance into specific crops (Kindle et al., 1999; Hain et al., 1998; Schroder et al., 2000).

In relation to the antimicrobial activity of pine needle extract, there have been studies to develop antimicrobial material against food rot by using extract. However, the direct extraction of the stilbene compound to some or all of the extract as an antifungal agent, fungicide, antimicrobial agent or the like, or synthesized stilbene compound present in pine needles and used as the antifungal agent, fungicide, antimicrobial agent There was no precedent.

Accordingly, it is an object of the present invention to provide a method for synthesizing the stilbene compound parent in pine needles and mass production in view of the above facts.

Another object of the present invention is to assay the antifungal and antibacterial function of the synthesized pine needle stilbene compound.

Still another object of the present invention is to prepare an antimicrobial agent, fungicide material and food preservative using a composition containing the pine needle stilbene compound has been confirmed antifungal and antibacterial function.

The above object of the present invention was achieved by synthesizing a large amount of stilbene compound pinosylbin in pine needles, and assaying the antifungal and antibacterial function of the synthesized pinosylbin, the effect on the shelf life of fruit and bactericidal ability.

Hereinafter, it demonstrates concretely in the structure of this invention.

1 is a result of NMR analysis of the structure and purity of stilbene-based pinosylbin in pine needle extract components.

2 is a result of confirming the antimicrobial and antifungal efficacy of the microorganism (a) and the fungus (b) generated from the decayed tangerine, which is a stilbene compound of the present invention synthesized in the air, by a paper disk method.

Figure 3 shows the antimicrobial efficacy of the synthesized stilbene compound of the present invention, the bacterium bacillus Bacillus cereus (a) and Staphylococcus aureus (b) This is the result confirmed by the method.

Figure 4 is a result of confirming the antifungal effect on the fungal infection of the stilbene compound of the present invention synthesized pinosylbin to fruit and disease.

5 is a result of confirming the antimicrobial efficacy against microorganisms in the air after treatment for 15 minutes at 121 ℃ in order to confirm the thermal stability of the synthesized stilbene-based compound of the present invention.

Figure 6 shows the bactericidal efficacy of the bacterium stilbene compound of the present invention synthesized the bacterium Bacillus cereus (a) and Staphylococcus aureus (b) causing food poisoning This is the result of checking.

The present invention comprises the steps of synthesizing pinosylbin stilbene compound in pine needles; It consists of the antifungal and antibacterial function of the synthesized pinosylbin, the effect on the shelf life of the fruit and the bactericidal activity.

The method for preparing pinosylbin, the stilbene-based compound of the pine needles of the present invention, is characterized by including the following steps:

TLC the mixture of benzylbromide and triethyl phosphite, remove excess triethylphosphite through distillation to obtain diethylbenzylphosphonate,

After cooling the mixture of diethylbenzylphosphonate obtained above and dried dimethylformamide and sodium methoxide under nitrogen gas, 3,5-dimethoxybenzyl aldehyde was added to the mixture, followed by stirring at room temperature for overnight. Politics,

TLC the mixture and add a mixture of water and methanol to precipitate the reaction mixture,

The product obtained above is dried, the filtrate is purified by adsorption column chromatography to obtain 3,5-Dimethoxystilbene,

3,5-dimethoxystilbene and pyridine hydrochloride were mixed under nitrogen gas, stirred in an oil bath at 190 ° C., followed by TLC, and then 2N HCl was injected and ether solution (5 Extract × 5 mL), and

Water is removed from the ether layer resulting from the above, filtered and the solvent of the filtrate is removed by evaporation,

The product obtained therefrom was purified by column chromatography.

The structure of the pinocylbin in the stilbene compound in the pine needle of the present invention is represented by the following formula (I):

The antimicrobial activity of the pinocylbin derived from pine needles of the present invention was evaluated through a paper disk test and a minimum inhibitory concentration (MIC) test.

In the present invention, Staphylococcus aureus and Bacillus cereus were used as food poisoning related bacteria used to test the antimicrobial activity of pinocylbin derived from pine needles. The Bacillus cereus ( B. cereus ) was used KCTC 1012 strain that was distributed from the gene bank in the Korea Institute of Bioscience and Biotechnology Genetic Center, Staphylococcus aureus ( A. American Type Culture Collection) ATCC 25923 strain sold at

The microorganisms in the air used in the embodiment of the present invention was selected as a colony (colony) of the microorganisms generated in the nutrient agar plate that was left in the air and used again as a test strain.

Hereinafter, specific examples of the present invention will be described in detail with reference to Examples, but the scope of the present invention is not limited thereto.

EXAMPLE

Example 1: Preparation of pinosylbin, a pine needle-derived development agent

In order to synthesize the pinocylbin in stilbene compounds in the pine needle of the present invention, 4.3 mL of benzyl bromide and 10 mL of triethyl phosphite were added to a 25 mL-round bottomed bottle. The mixture was refluxed with stirring at n ° C., and n-hexane and ethyl acetate were mixed at 3: 7 (v: v) as a developing solvent to observe the progress of TLC (Thin Layer Chromatography) reaction. After the development of ethyl bromide was completed, excess triethylphosphite was removed by distillation using a vacuum trap to obtain diethylbenzylphosphonate.

Then, diethylbenzylphosphonate obtained above and dried dimethylformamide (50 mL) were injected into a 100 mL-round bottomed bottle under nitrogen gas using a syringe, sodium methoxide (2 g). After the addition, the mixture was stirred until cooled in an ice bath (Ice bath, 0 ° C.), and after cooling at 0 ° C., 3,5-dimethoxybenzaldehyde (4 g) was added thereto, followed by stirring for about 15 minutes. After stirring for 1 hour at room temperature and stirring with a steam bath (steambath, 100 ℃), after 5 hours left to stand at room temperature overnight.

N-hexane and ethyl acetate were mixed to 8: 2 (v: v) as a developing solvent, and TLC reaction was observed. After completion of the reaction, water: methanol (2: 1, 80 mL) was added to the reaction mixture. Precipitation was observed. After precipitation, paper glass filtration was performed with water and the product was dried.

The filtrate was evaporated in a reduced pressure evaporator, and the solvent was evaporated sufficiently using a vacuum trap. The product obtained therefrom was developed using a developing solvent having a mixing ratio of 96: 4 (v: v) of n-hexane and ethyl acetate. Purification by adsorption column chromatography. NMR conditions are as follows:

¹ H-NMR (DMSO-d _6, 200 MHz), δ 3.78 (s, 6H, OMe), δ6.44 (t, 1H, H-4), δ6.81 (d, 2H, H-2 & H -6), δ7.23 (AB system, 2H, J = 8 Hz, CH = CH), δ 7.36 (m, 3H, H-3 ', H-4'& H-5 '), δ7.6 ( d, 2H, J = 6 Hz, H-2 '&H-6').

The purified 3,5-dimethoxystilbene (2g) and the excess of pyridine hydrochloride (10g) was added to a 100 mL-round bottomed bottle under nitrogen gas and oil bath ( The mixture was refluxed at 190 ° C. with an oil bath and mixed with n-hexane and ethyl acetate as 1: 1 (v: v) as a developing solvent, and the progress of TLC reaction was observed. When the reaction was completed, 2N HCl was injected into the syringe, stirred slowly for 10 minutes, transferred to a separatory funnel, and extracted using an ether solution (5 × 5 mL). Thereafter, the ether layer was removed with water using magnesium sulfate, filtered, and the filtrate was evaporated by a dry evaporator under reduced pressure, and then the solvent was completely removed using a vacuum trap. The product obtained therefrom was purified by column chromatography using a developing solvent having a mixing ratio of 8: 2 (v: v) of n-hexane and ethyl acetate, and crystallized with benzene to obtain a high purity product. NMR conditions are as follows:

¹ H-NMR (DMSO-d _6, 200 MHz), δ6.15 (s, 1H, H-4), δ6.45 (d, 2H,, J = 6Hz H-2 & H-6), δ7.04 (AB system, 2H, CH = CH), δ 7.36 (m, 3H, H-3 ', H-4'& H-5 '), δ 7.5 (d, 2H, J = 6 Hz, H-2 '&H-6'), δ 9.28 (s, 2H, -OH).

Figure 1 shows the results of analyzing the structure and purity of the pinosylbin by NMR, the structure of the pinosylbin is shown in the following formula (I):

Example 2: Evaluation of antimicrobial activity through paper disk test of pinotsilbin, a development material of pine needles

The antibacterial activity against the fungus causing the decay of the bacteria or fruits (tangerines) of the pine needles derived from pine needles of the present invention was evaluated using a paper disk test.

Pinocylbin, a pine needle-derived development agent, was dissolved in ethanol, and 0.1-1 mg / mL concentration was absorbed by dropping on an 8 mm paper disc and used for decaying bacteria. For testing for microorganisms in the air was treated with pinosylbin concentration of 0.01 ~ 0.1 mg / mL. The microorganisms in the air were obtained by selecting one colony (colony) among the microorganisms generated in the nutrient agar plate that was left in the air and cultured again. The test strain and the fungal fungal liquid of the decayed tangerine were placed in a nutrient agar plate, which was incubated at 30 ° C. for 24 hours, and then evaluated for the antimicrobial activity by measuring the presence and diameter of the growth zone around the disc. It was. Ethanol was treated and used as a control.

As shown in FIG. 2, pinosylbin showed antimicrobial activity in a concentration-dependent manner against both microorganisms and fruit rot bacteria in air, and at higher concentrations against mold. In the paper disk test, the concentration range of pinosylbin showing antimicrobial activity was 0.5 to 1 mg, and no growth inhibition zone was observed in ethanol used as a control.

Example 3: Evaluation of antimicrobial activity through MIC (Minimum Inhibitory Concentration) test of pinocylbin, a pine needle-derived development agent

The antimicrobial activity of the pinocylbine derived from pine needles of the present invention against airborne microorganisms and food poisoning related bacteria was evaluated using a minimum inhibitory concentration (MIC) test.

① Evaluation of antimicrobial activity against airborne microorganisms

One colony was selected from the microorganisms generated in the nutrient agar plate, which was left in the air, and then cultured again, and used as a test strain. Agar plates (nutrient agar) were prepared to contain pinosylbin in the concentration range of 0.01 mg / mL to 0.1 mg / mL to inoculate the test strain. After incubation at 30 ° C. for 24 hours, the concentration was observed by visual observation to determine the concentration of the microorganisms by MIC.

② Evaluation of antimicrobial activity against Bacillus cereus (KCTC 1012)

A test tube containing MHB (Muller Hinton Broth) medium containing 5 mg / mL of pinocylbin dissolved in ethanol at 0.2%, 0.39%, 0.79%, 1.56%, and 3.13%, respectively, was prepared. The test tube was inoculated with the strain and incubated at 22 ± 2 hours at 35 ± 0.5 ° C. to determine the turbidity of the media in each test tube, and the concentration of the test tube without bacterial growth was determined by MIC.

③ Evaluation of antimicrobial activity against Staphylococcus aureus (S taphylococcus aureus , ATCC 25923)

A test tube containing MHB (Muller Hinton Broth) medium containing 5 mg / mL of pinosylbin dissolved in ethanol at 0.2%, 0.39%, 0.78%, 1.56%, and 3.13%, respectively, was prepared. The test tube was inoculated with the strain and incubated at 22 ± 2 hours at 35 ± 0.5 ° C. to determine the turbidity of the media in each test tube, and the concentration of the test tube without bacterial growth was determined by MIC.

MIC for each strain Strain name MIC Remarks Microbes in the air 0.03-0.1 mg / mL Antibacterial activity is maintained even after heat treatment at 12.1 ℃ for 15 minutes B. cereus KCTC 1012 0.078 mg / mL Food Poisoning Bacteria Staphylococcus aureus ( S. aureus ATCC 25923) 0.078 mg / mL Food Poisoning Bacteria

As shown in Table 1 and Figure 3, MIC test results showed that pinosylbin showed antimicrobial activity at concentrations of 0.78 mg / mL or more against staphylococcus aureus and Bacillus cereus, bacteria associated with food poisoning.

Example 4 Effect on the Shelf-life of Fruit (Tanger) of the Invention

The present invention was tested for the shelf life of the fruit by treating the pinocylbin to the tangerine. Water, alcohol, and pinosylbin (0.1 mg / mL of pinosylbin dissolved in ethanol) were applied to tangerines, respectively, and the degree of decay of the microorganisms was confirmed as time passed. On the 11th day, photographs of the tangerines were compared and observed for each treatment group, and the front and rear views of the tangerines were checked until after one month.

As shown in FIG. 4, in the case of the tangerine-treated tangerine, freshness was maintained for at least one month.

Example 5 Thermal Stability Test of the Invention Pinocylbins

In order to confirm the thermal stability of the pinocylbin of the present invention, the amino plate inoculated with the microorganisms in the air prepared according to Example 3 was treated with pinocylbin at a concentration of 10 μg / mL to 100 μg / mL at 121 ° C. for 15 minutes. The activity of silvin and untreated pinosylbin was compared.

As shown in FIG. 5, it was confirmed that the pinocylbin was relatively stable even in heat because it was active even after treatment at 121 ° C. for 15 minutes.

Example 6: Evaluation of bactericidal power of pinosylbin, a pine needle-derived development agent

In order to evaluate the bactericidal power of the pine needle-derived development agent, pinocylbin, Bacillus cereus (KCTC 1012) and Staphylococcus aureus (ATCC 25923), which are food poisoning-related bacteria, were cultured and then cultured. The bacteria were diluted and used to test the initial inoculum to be 1-9 x 10 ⁵ cfu / mL. Pinosylbin was added at 0.4 mg / mL, and each cultured bacterium was inoculated into agar plate, and then 10 minutes and 30 minutes, the number of bacteria was measured and the reduction rate was calculated. The number of bacteria was calculated by multiplying the number of bacteria on the medium by the dilution factor, and the reduction rate was calculated as follows.

(However, A: the initial number of bacteria, B: the number of bacteria after a certain time)

As shown in FIG. 6, pinosylbin showed the effect of sterilizing more than 96% within 10 minutes at a concentration of 0.4 mg / mL for bacteria associated with food poisoning, 99.9% after 30 minutes. In the bactericidal effect, Staphylococcus aureus showed a more sensitive reaction than B. cereus .

Number of bacteria by time (unit: CFU / mL) Strain name Early 10 minutes later 30 minutes later B. cereus 1 x 10 ⁵ 4.2 x 10 ² <10 Staphylococcus aureus 1.2 x 10 ⁵ <10 <10

% Bacterial reduction over time Strain name 10 minutes later 30 minutes later B. cereus 99.6 99.9 Staphylococcus aureus 99.9 99.9

As described above through the above examples, the present invention relates to an antimicrobial composition containing a pinocylbin which is a stilbene-based compound among pine needle extract components and a method for synthesizing the pinosylbin, which is a stilbene-based compound among pine needle components There is an excellent effect in providing a method for synthesizing pinosylbin in large quantities. In addition, the pinocylbin of the present invention has an antifungal and antibacterial function, so that the composition containing the compound as an active ingredient is a very useful invention for the functional fine chemical products, food, and cosmetic industries.

Claims (6)

TLC the mixture of benzylbromide and triethyl phosphite, remove excess triethylphosphite through distillation to obtain diethylbenzylphosphonate, After cooling the mixture of diethylbenzylphosphonate obtained above and dried dimethylformamide and sodium methoxide under nitrogen gas, 3,5-dimethoxybenzyl aldehyde was added to the mixture, followed by stirring at room temperature for overnight. Politics, TLC the mixture and add a mixture of water and methanol to precipitate the reaction mixture, The product obtained above is dried, the filtrate is purified by adsorption column chromatography to obtain 3,5-Dimethoxystilbene, 3,5-dimethoxystilbene and pyridine hydrochloride were mixed under nitrogen gas, stirred in an oil bath at 190 ° C., followed by TLC, and then 2N HCl was injected and ether solution (5 Extract × 5 mL), and Water is removed from the ether layer resulting from the above, filtered and the solvent of the filtrate is removed by evaporation, A method for synthesizing pinosylbin, which is a stilbene-based compound of pine needles, which comprises purifying a product obtained by column chromatography. The method for synthesizing pinosylbin which is a pine needle-derived stilbene compound according to claim 1, wherein a mixture of n-hexane and ethyl acetate is used as a developing solvent for carrying out the TLC. Pinosylvinyl of formula (I) An antifungal composition comprising the pinocylbin of claim 3 as an active ingredient. An antimicrobial composition comprising the pinocylbin of claim 3 as an active ingredient. A composition for household goods and food preservatives, comprising the pinocylbin of claim 3 as an active ingredient.