KR100280368B1 - Natural Antibacterial Components Isolated from the Bark of Pine Trees - Google Patents

Abstract

The present invention relates to a natural antimicrobial component isolated from the bark of pines, and more particularly, after extracting the bark of pines discarded in the existing timber industry with a mixed solution of methanol and water, washing and fractionation using several organic solvents. It relates to a natural antimicrobial component excellent in antibacterial activity obtained by the process.

Description

Natural Antibacterial Components Isolated from the Bark of Pine Trees

The present invention relates to a natural antimicrobial component isolated from the bark of pines, and more particularly, after extracting the bark of pines discarded in the existing timber industry with a mixed solution of methanol and water, washing and fractionation using several organic solvents. It relates to a natural antimicrobial component excellent in antibacterial activity obtained by the process.

As a method of using a natural product extracted from a plant as an antimicrobial substance, the following methods are known. For example, a method of separating and purifying the water-soluble layer having antimicrobial activity by extracting the residue remaining after adding a nonpolar solvent to the ground poongyoung dry powder pulverized [Korea Patent Publication No. 95-5739], mugwort extract Method for preparing soap by blending green tea extract in order to increase the antimicrobial activity to the soap [Korean Patent Publication No. 95-10612], the method using the antibacterial properties of grapefruit seed extract [Korean Patent Publication Nos. 93-009496 and 96- 3619].

In addition, as a method of using the extract of pines, a method of using a soap composition containing pine needle components [Korean Patent Publication No. 95-7720], a pine needle extract having a role of promoting skin aging and blood circulation as a cosmetic soap composition [ Republic of Korea Patent Application Publication No. 93-23450, Method of producing noodles using pine and pine needles [Korea Patent Publication No. 97-32453], Method of using pine shoot extract as a beverage composition [Korea Patent Publication No. 97-32508], Method of using pine extract as an anti-hair loss and hair growth promoting agent by using the vasodilatation effect of essential oil which is a pine extract [Korean Patent No. 96-40346], by adding an extract of pine leaves, sprouts and eggplant to shampoo or rinse There is a method for maintaining the cleanliness and hygiene of the scalp (Korean Patent Publication No. 94-10987).

However, there is no example of using an extract derived from pine bark as an antibacterial substance.

Therefore, the present invention is to provide a natural antibacterial component having excellent antimicrobial activity by applying a simple extraction and separation method using the pine bark as a material.

1 is a process diagram schematically showing a process of extracting the antimicrobial component from the bark of pines according to the present invention,

Figure 2 is a process diagram schematically showing a method for separating the antimicrobial component through a solvent fractionation process from the bark extract obtained through the process of FIG.

The present invention is characterized by a natural antimicrobial component obtained by crushing the pine bark, extracted with a mixed solution of water and methanol, and concentrated, and then washed and fractionated using a different organic solvent for each polarity.

Referring to the present invention in more detail as follows.

The present invention extracts the antimicrobial component of the bark of pines discarded as waste in the wood industry with a mixed solution of water and methanol, and simply washes and fractionates them by polarity using several organic solvents. To get.

As the pines used in the present invention, for example, one or two or more selected from red pine, ridged pine, and radiata pine can be suitably used. Such pines can be used as waste produced in the wood industry. In order to make it easy to penetrate and to make it suitable for extraction, it is used to grind it in 80-100 mesh size.

In addition, in the present invention, a mixed solution of water and methanol is used for initial solvent extraction, and the mixing ratio thereof is good in terms of complete extraction of the active ingredient using a mixture of methanol: water in a volume ratio of 5: 1 to 1: 5. .

On the other hand, the present invention is an important feature to greatly improve the extraction effect by the polar washing method to sequentially wash the extract using different organic solvents for the separation of effective antibacterial components, solvent extraction for each polarity is for example The antimicrobial component can be effectively separated by degreasing with heptane followed by rinsing with diethyl ether and similarly polar ethyl acetate and butanol. At this time, degreasing with heptane is for removing fat-soluble components, and for effective degreasing, it is preferable to use after dissolving the extract before use of heptane in methanol.

The extraction and separation method according to the present invention will be described in more detail in a series of processes with reference to FIGS. 1 and 2 as follows.

Extracting the bark from the bark, as shown in Figure 1 bark obtained from pines (Red pine, Rigida pine and Radiata pine) as the solvent easily penetrates the size of about 80 to 100 mesh and then the raw material is methanol: Water was immersed in the mixed solution mixed at a volume ratio of 5: 1 to 1: 5, left for several days, and then filtered to obtain an extract. Extraction at this time can be carried out repeatedly several times to increase the yield of the extract. The extracts thus obtained are combined and concentrated to about 1/10 of the original volume using a reduced pressure concentrator. In this state, mostly water is present.

The concentrated extract is washed with a process as shown in FIG. 2. First, methanol is added to completely dissolve all components, and then heptane is added to perform degreasing through layer separation, which is also repeated several times. Then, diethyl ether was added to the concentrated heptane washed solution, followed by washing several times with fractions to remove nonpolar components, followed by several times using ethyl acetate in the same manner. The continuous washing and fractionation of diethyl ether and ethyl acetate is a method that has not been tried in the existing separation methods because the properties of the two solvents are similar. According to the results of the present invention it was found that the continuous use of such diethyl ether and ethyl acetate is an important factor that can increase the antimicrobial activity compared to the conventional method using only one of these two solvents. In addition, the present invention is extracted again with butanol after the continuous extraction of the diethyl ether and ethyl acetate in order to separate the polar material contained in the water layer.

The fractions thus obtained are concentrated and spray-dried to complete the commercialization of natural antibacterial components.

As a result of performing the antimicrobial activity test on the natural extract of the present invention as described above, it can be seen that it has excellent antimicrobial activity against not only food microorganism but also paper mill slime forming bacteria.

Hereinafter, the present invention will be described in more detail based on the following examples, but the present invention is not limited thereto.

Example 1

The bark obtained from the 15-year-old red pine collected as a sample was ground to a size of about 80 to 100 mesh, and then the crushed sample was immersed in 10 times of methanol: water (3: 1, v / v) mixed solution and then 2-3. After standing for one day, the solution was collected by filtration. In order to increase the yield of the extract was again replaced with a new methanol: water mixture solution, and the extraction was left for 2 to 3 more days, and then filtered again to obtain an extract. The obtained extracts were combined and concentrated to about 1/10 of the original volume using a vacuum concentrator. In this state, mostly water is present.

The concentrated extract was added with the same amount of methanol to completely dissolve all the components, and then shaken vigorously by adding the same amount of heptane, followed by standing. Thereafter, when the layer was formed, degreasing was performed by removing the heptane layer, and this heptane washing process was repeated twice (preparation of heptane fraction). An equal amount of diethyl ether (Et ₂ O) was poured into the concentrated solution of heptane washing, and washing fractions were carried out three times in the same manner as in the case of heptane to remove nonpolar components (Et ₂ O fraction preparation). For a more thorough wash fraction, an equal amount of ethyl acetate (EA) having a polarity similar to diethyl ether was poured into the concentrate and washed three times in the same manner as above (EA fraction preparation). And butanol was tried three times in the same manner as described above (butanol fraction preparation). Each fraction thus obtained was concentrated and then dried using a spray dryer to powder.

Examples 2 to 3

The same procedure as in Example 1 was carried out, but the 15-year-old Rigida pine and radiata pine were used as samples.

Experimental Example

The results of measuring the antimicrobial activity of the bark extract prepared in Examples 1 to 3 are as shown in Table 1.

-Antimicrobial activity measurement method-

The test bacteria were stored in a deep freezer in a container containing a mixture of glycerol and LB broth (Luria-Bertani; tryptone 1g, yeast extract 0.5g, NaCl 0.5g, glucose 0.1g, distilled water 100ml). . The stock solution was taken in an appropriate amount and cultured in a fresh LB culture until the maximum growth of bacteria. The spores were taken from the culture medium, and added in a 10 ml LB medium so that the concentration of the spores was 10 ^6. Then, the extract fractions obtained in Examples 1 to 3 were prepared at 200 ppm, 400 ppm, 600 ppm, and 1000 ppm. Each maximum absorbance was measured at 610 nm after 24 hours of incubation. In the untreated plots were cultured without the pine bark extract, the bacterial growth inhibition was calculated by the following equation 1 compared to the untreated plots.

The antimicrobial activity (bacterial growth inhibition rate) of red pine bark extract fraction, Rigida pine bark extract fraction, and Radiata pine bark extract fraction based on the amount of 400 ppm added of each extract fraction is shown in Table 1, Table 2 and Table 3 below.

In addition, MIC (minimum inhibition concentration) of the test strains of each bark extract obtained as a result of this experiment are shown in Tables 4, 5 and 6, respectively.

Growth Inhibition Rate of Red Pine Bark Extract (%) Treatment concentration per extract fraction 400 ppm Heptane fraction Et ₂ O Fraction EA fraction Butanol fraction Escherichia coli 35 38 29 25 Aerobacter aerogenes 86 100 42 36 Pseudomonas aeruginosa 40 46 30 21 Klebsiella oxytoca 36 38 29 26 Erwinia aroideae 87 37 55 49 Proteus vulgaris 32 35 32 79 Pseudomonas fluorescens 34 38 32 76 Alcaligenes eutrophus 26 29 31 22 Bacillus subtilis 89 56 42 33 Staphylococcus aureus 89 49 35 19 Micrococcus flavus 79 39 35 30 Micrococcus luteus 84 46 45 89 Candida albicans 46 52 46 42 Saccharomyces acidofaciens 43 47 49 36 Saccharomyces diastaticus 50 59 50 38

Growth Inhibition Rate of Rigidasong Bark Extract (%) Treatment concentration per extract fraction 400 ppm Heptane fraction Et ₂ O Fraction EA fraction Butanol fraction Escherichia coli 35 28 29 22 Aerobacter aerogenes 100 46 45 32 Pseudomonas aeruginosa 42 39 36 29 Klebsiella oxytoca 100 64 48 49 Erwinia aroideae 84 42 48 32 Proteus vulgaris 34 36 25 24 Pseudomonas fluorescens 38 35 29 22 Alcaligenes eutrophus 33 35 19 16 Bacillus subtilis 100 53 48 22 Staphylococcus aureus 39 44 100 28 Micrococcus flavus 100 56 92 43 Micrococcus luteus 94 67 100 47 Candida albicans 57 56 48 32 Saccharomyces acidofaciens 70 78 49 39 Saccharomyces diastaticus 73 83 44 35

Growth Inhibition Rate of Radiata Songk Bark Extract (%) Treatment concentration per extract fraction 400 ppm Heptane fraction Et ₂ O Fraction EA fraction Butanol fraction Escherichia coli 49 36 90 33 Aerobacter aerogenes 100 100 68 45 Pseudomonas aeruginosa 42 46 78 39 Klebsiella oxytoca 100 100 89 42 Erwinia aroideae 88 100 90 54 Proteus vulgaris 68 100 56 39 Pseudomonas fluorescens 48 100 75 41 Alcaligenes eutrophus 42 45 65 39 Bacillus subtilis 85 76 88 46 Staphylococcus aureus 48 100 86 46 Micrococcus flavus 100 100 100 51 Micrococcus luteus 43 100 56 38 Candida albicans 85 79 55 36 Saccharomyces acidofaciens 90 82 62 47 Saccharomyces diastaticus 90 88 60 45

MIC of Testosterone Extracts of Red Pine Bark Extract MIC by extract fraction Heptane fraction Et ₂ O Fraction EA fraction Butanol fraction Escherichia coli - - - - Aerobacter aerogenes 600 400 - - Pseudomonas aeruginosa - - - - Klebsiella oxytoca - - - - Erwinia aroideae 600 - - - Proteus vulgaris - - - 1000 Pseudomonas fluorescens - - - 1000 Alcaligenes eutrophus - - - - Bacillus subtilis 600 - - - Staphylococcus aureus 600 - - - Micrococcus flavus 1000 - - - Micrococcus luteus 600 - - 1000 Candida albicans - - - - Saccharomyces acidofaciens - - - - Saccharomyces diastaticus - - - - -: No inhibition

MIC of Species Strains of Rigidasong Bark Extract MIC by extract fraction Heptane fraction Et ₂ O Fraction EA fraction Butanol fraction Escherichia coli - - - - Aerobacter aerogenes 400 - - - Pseudomonas aeruginosa - - - - Klebsiella oxytoca 400 1000 - - Erwinia aroideae 600 - - - Proteus vulgaris - - - - Pseudomonas fluorescens - - - - Alcaligenes eutrophus - - - - Bacillus subtilis 400 - - - Staphylococcus aureus - - 400 - Micrococcus flavus 400 - 600 - Micrococcus luteus 600 1000 400 - Candida albicans - - - - Saccharomyces acidofaciens 1000 1000 - - Saccharomyces diastaticus 1000 600 - - -: No inhibition

MIC of Different Radical Songk bark Extracts MIC by extract fraction Heptane fraction Et ₂ O Fraction EA fraction Butanol fraction Escherichia coli - - 600 - Aerobacter aerogenes 400 400 1000 - Pseudomonas aeruginosa - - 1000 - Klebsiella oxytoca 400 400 600 - Erwinia aroideae 600 400 600 - Proteus vulgaris 1000 400 - - Pseudomonas fluorescens - 400 1000 - Alcaligenes eutrophus - - 1000 - Bacillus subtilis 600 1000 600 - Staphylococcus aureus - 400 600 - Micrococcus flavus 400 400 400 - Micrococcus luteus - 400 - - Candida albicans 600 1000 - - Saccharomyces acidofaciens 600 600 1000 - Saccharomyces diastaticus 600 600 1000 - -: No inhibition

As described above, the natural antimicrobial component extracted from the pine bark according to the present invention has excellent antimicrobial activity against food microorganisms as well as paper mill slime forming bacteria.

Claims (5)

In the method of extracting and separating antimicrobial components, the wood components are pulverized, extracted with a mixed solution of water and methanol, concentrated, and then the concentrated solutions are sequentially washed and fractionated with different organic solvents for each polarity to obtain a natural extract. The bark of pines discarded as wood components and the sequential washing using the organic solvent for each polarity are antibacterial from the bark of pine trees, characterized by sequentially using diethyl ether, ethyl acetate and butanol after degreasing with heptane. How to extract and separate ingredients. The method of claim 1, wherein the mixed solution of water and methanol is a method of extracting and separating antimicrobial components from the bark of pines, wherein methanol: water is mixed in a volume ratio of 5: 1 to 1: 5. The method of extracting and separating antibacterial components from the bark of a pine tree according to claim 1, wherein the concentrate is sufficiently dissolved in methanol prior to degreasing to heptane. According to claim 1, wherein the pine bark is a method of extracting and separating the antibacterial components from the bark of pines, characterized in that the mixture of one or two or more selected from red pine, Rigida pine and radiata pine. Natural antibacterial component from pine bark, characterized in that it is extracted and separated according to claim 1.

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