WO2014193031A1

WO2014193031A1 - Antimicrobial agent having moringa oleifera extract captured in porous zinc oxide and preparation method therefor

Info

Publication number: WO2014193031A1
Application number: PCT/KR2013/006695
Authority: WO
Inventors: 이근하; 권순상; 공혜진; 박청; 황재성; 신동하
Original assignee: (주)모아캠; 경희대학교 산학협력단
Priority date: 2013-05-31
Filing date: 2013-07-26
Publication date: 2014-12-04
Also published as: KR101452326B1

Abstract

The present invention relates to an antimicrobial agent having a Moringa oleifera extract captured in a porous material and a preparation method therefor and, more particularly, to an antimicrobial agent prepared by capturing a Moringa oleifera seed extract in zinc oxide which is a porous material. The purpose of the present invention is to provide the antimicrobial agent which is harmless to the human body, on the basis of the porous material and the Moringa oleifera seed extract. In order to achieve the purpose, the present invention provides the antimicrobial agent having the Moringa oleifera seed extract captured in the zinc oxide (ZnO) which is a porous material, and the preparation method therefor.

Description

Antibacterial agent containing Moringa oleifera extract in porous zinc oxide and its preparation method

The present invention relates to an antimicrobial agent in which Moringa oleifera extract is collected in a porous material, and more particularly, to an antimicrobial agent prepared by collecting Moringa oleifera extract in a zinc oxide, which is a porous material.

Cosmetics are contaminated with various pathogens due to repeated contact with hands. To prevent this, a strong antibacterial substance is added to the cosmetics.

Antimicrobial agents are roughly classified into organic antimicrobial agents and inorganic antimicrobial agents. Organic antimicrobial agents include surfactants, biguanides, and alcohols, and inorganic antimicrobial agents include zeolite, silica gel, glass, calcium phosphate, zirconium phosphate, calcium silicate, BACKGROUND ART Antibacterial agents in which inorganic powders such as titanium oxide, zinc oxide, silicon dioxide and alumina are supported on an antimicrobial metal such as silver (Ag), copper (Cu) and zinc (Zn) are known.

Inorganic antimicrobial agents have the advantage of having good antimicrobial properties for a long time and excellent heat resistance since there is no volatilization or decomposition compared to organic antimicrobial agents.

In general, chemical antibacterial ingredients such as copper, zinc or metals such as parabens are used. Antibacterial metals such as copper or zinc can be harmful to the human body, and their use is regulated, particularly for inorganic zinc.

In order to overcome this problem, Korean Laid-Open Patent Publication No. 10-2009-0096799 discloses a method for preparing a fungicide using silver. However, such an inorganic antimicrobial agent has a disadvantage in that antimicrobial activity is less effective than organic antimicrobial agents, and a large amount of antimicrobial agent is required to obtain antimicrobial anticipation, which is expensive compared to organic antimicrobial agents.

In addition, with respect to the antimicrobial effect of natural plant extracts, Korean Patent Publication No. 10-1221735 discloses the antimicrobial effect of Syzygium aromaticum (clove), Cinnamomum japonicum sieb ( Cinnamomum japonicum sieb) It is known about the antimicrobial effect of extracts from wild grapes, but only natural plant extracts have a problem that the antimicrobial effect against microorganisms such as Escherichia coli is not excellent (Han Jung-hoon, Master's degree in forestry engineering, Chonbuk National University) Antimicrobial and antibacterial effects, 2011).

On the other hand, Japanese Patent Laid-Open No. 2000-229804 discloses an antimicrobial agent containing Moringa oleifera extract as an active ingredient, but the present invention is different in that the porous material in which the Moringa oleifera extract is collected is used as an antimicrobial agent.

Accordingly, the present inventors have made diligent efforts to solve the above problems, confirming that the antimicrobial agent in which Moringa oleifera seed extract is collected in the porous material has an excellent antimicrobial effect while harmless to the human body as an organic-inorganic compound, and the present invention To complete.

Summary of the Invention

An object of the present invention is to provide an antimicrobial agent harmless to the human body based on the porous material and Moringa oleifera seed extract. In order to achieve the above object, the present invention provides an antimicrobial agent and a method for preparing the moringa oleifera extract is collected in a zinc oxide (ZnO) is a porous material.

Figure 1 shows the XRD analysis of the porous zinc oxide particles by the sol-gel method.

Figure 2 shows the SEM analysis of the porous zinc oxide particles.

Figure 3 shows the results of TEM analysis of porous zinc oxide particles.

Figure 4 shows the chemical structure of Niazimicin.

Figure 5 shows the chemical structure of 4- (α-L-rhamnopyranosyloxyl) -benzyl isothiocyanate (RBI).

Figure 6 shows the antimicrobial effect test results by the circular filter method, A is the experimental results of Niazimicin, B is the experimental results of 4- (α-L-rhamnopyranosyloxyl) -benzyl isothiocyanate (RBI).

Detailed Description of the Invention and Specific Embodiments

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In general, the nomenclature used herein is well known and commonly used in the art.

In one embodiment of the present invention, the antimicrobial effect was confirmed by preparing an antimicrobial agent to collect the Moringa oleifera extract extract in zinc oxide (ZnO), a porous material. As a result, it was confirmed that there is an antimicrobial effect against microorganisms such as Staphylococcus aureus and Escherichia coli.

The term "antimicrobial agent" in the present invention means a substance that inhibits the production and activity of microorganisms or bacteria or kills microorganisms and bacteria, but is not limited thereto.

Moringa oleifera extract of the present invention may be prepared according to a method for producing a variety of plant extracts known in the art, for example, may be by heat extraction, reflux extraction or heat extraction, but is not limited thereto.

The Moringa oleifera extract may be an extract extracted with water or an organic solvent, ethanol, hexane, chloroform, ethyl acetate, methanol, propanol, low cost alcohol having 1 to 4 carbon atoms, etc. may be used as the organic solvent, but is not limited thereto. It is not.

Therefore, the present invention relates to an antimicrobial agent in which Moringa oleifera extract is collected in a porous material.

In the present invention, the porous material may be characterized in that the zinc oxide (ZnO), the Moringa oleifera extract may be characterized in that the Moringa oleifera seed extract. The Moringa oleifera seed extract is preferably using Moringa oleifera seed ethanol extract or Moringa oleifera seed ethyl acetate extract.

In another aspect, the present invention relates to a method for producing an antimicrobial agent, which comprises collecting Moringa oleifera extract in a porous material.

In the present invention, an initial wet method may be used as a method for collecting the Moringa oleifera extract in the porous material, but is not limited thereto.

Example

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention, and it will be apparent to those skilled in the art that the scope of the present invention is not to be construed as being limited by these examples. Thus, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

Example 1 Preparation of Antimicrobial Agents

1-1: Preparation of Porous Zinc Oxide (ZnO) Particles

Porous zinc oxide was synthesized using a precipitation method in a polyol solvent. In order to easily control the particle size, sodium hydroxide was added. The molar ratio of zinc acetate dihydrate (Zn (CH ₃ COO) ₂ .2H ₂ O, Mw = 219.5, SAMJUN, Korea), which is a precursor of zinc oxide, to sodium hydroxide was 1: 1. Diethylene glycol (C ₂ H ₁₀ O ₃ , Mw = 106.12, SAMJUN, Korea) was used as the solvent. The reaction mixture was mixed under reflux conditions of 443 K. After the reaction was completed, the precipitate was rinsed with ethanol and centrifuged at 10,000 rpm to recover the porous ZnO particles.

The XRD pattern of the zinc oxide particles prepared in 1-1 was used as the standard of Joint Committee on Powder Diffraction Standards (JCPDS, 36-1451, a = 3.429 Å, c = 5.206 Å, λ = Cu 1.54 Å) Compared with data. Characteristic peaks of the ten zinc oxide particles appeared, which are (100), (002), (101), (102), (110), (103), (200), (112), (201) of zinc oxide. ), (004) and (202) is consistent with the decision aspect (Fig. 1).

The surface area and pore size of the zinc oxide particles prepared in 1-1 were measured by the Brunaure-Emmett-Teller (BET) instrument (Tristar, Micrometrics, USA) and the BJH (Barrett-Joyner-Halenda) method.

The pore characteristics, including the specific surface area, pore volume, pore size distribution and average pore diameter of the BET of the zinc oxide particles, are represented by N ₂ absorption / separation isotherms. The structure of the prepared zinc oxide particles was found to be a Type IV curve of H4 type hysteresis curve. The range of P / P ₀ consistent with the hysteresis was 0.4-0.7 and 0.9 or greater. This means that the initial particles have a mid-range pore size and the second particles with aggregated particles have a void space between the particles resulting from the aggregation of the initial particles. The BET surface area of the porous zinc oxide particles was 55 m ² / g. In the N ₂ absorption / isolation isotherm analysis, the pore sizes were 3 nm and 15-30 nm and the pore volume was 0.169 cc / g.

The morphology of the zinc oxide particles was evaluated using a field emission scanning electron microscope (SEM, JSM-7401F, JEOL, Japan) and a trans emission microscope (TEM, 300Kv, JEM2100F, JEOL, Japan).

When looking at the SEM image of the zinc oxide particles, it was found that the shape of the synthesized particles was spherical and the particle size was uniformly about 100 nm (FIG. 2). In addition, when looking at the TEM image, it was found that the spherical zinc oxide particles were agglomerates composed of very small nanoparticles having a size of 10 nm, and also confirmed in the TEM image that the particle size was 100 nm as demonstrated in the SEM image. (FIG. 3).

1-2: Preparation of Moringa oleifera extract

100 g of dried Moringa oleifera seed was pulverized, stirred and extracted with 70% (v / v) ethanol aqueous solution for 3 days, and filtered through a filter paper having a permeation size of 1.2 μm to obtain a filtrate. The filtrate obtained was concentrated under reduced pressure and dried under reduced pressure at 50 ° C. or lower to obtain 8.3 g of Moringa oleifera seed extract.

On the other hand, to obtain the Moringa oleifera seed extract using ethyl acetate (Ethyl acetate) using an organic solvent (hexane), oil components from the concentrated powder reduced pressure using a polar / non-polar fraction method, an optional separation of the oil phase 10 times distilled water was dispersed in the extract powder, and the same amount of hexane was added, mixed several times, and left until the two phases of nonpolar and polar phases were separated from each other to form a hexane layer (nonpolar). Separated from.

The same amount of ethyl acetate was added to the water layer (polar) from which the oil component was removed, mixed several times, and left until the two phases were separated from each other to obtain an ethyl acetate layer. The ethyl acetate layer thus obtained was concentrated under reduced pressure and dried under reduced pressure at 50 ° C. or lower to obtain 4.5 g of Moringa oleifera extract.

Moringa oleiferid extract obtained from the ethyl acetate prepared above using a water: acetonitrile (92: 8 ~ 8:92, Gradient) in a C18 reversed phase column using prep HPLC or MPLC as a mobile phase Niazimicin at UV wavelength 256nm (FIG. 4) and 4- (α-L-rhamnopyranosyloxyl) -benzyl isothiocyanate (RBI) (FIG. 5) were separated into two bioactive materials.

1-3: Collection of organic material into porous zinc oxide (ZnO) particles

In order to capture the guest material in the porous zinc oxide particles, the mixture was mixed with 50% (w / v) organic material and rubbed to infiltrate the solution into the pores of the porous zinc oxide particles. In particular, in order to investigate the synergistic antimicrobial effect between inorganic zinc oxide and organic materials, the extract was selected from Moringa oleifera seed as a guest material in porous zinc oxide, and the collection efficiency in porous zinc oxide was applied to Moringa oleifera seed extract. 10% (w / w). The procedure was repeated three times to capture the Moringa oleifera extract in the porous zinc oxide channel.

Example 2: Evaluation of Characteristics of Prepared Antimicrobial Agents

All experiments were performed in triplicate. Data are presented as mean ± standard deviation (SD). Means were assessed by Student's multirange t-test. Statistically, the significant difference is when p <0.05.

The evaluation of the antimicrobial properties was made by measuring the paper disc method and the minimum inhibition concentration method (MIC).

Characterization of the antimicrobial agent by the paper disc method was performed by precipitating the circular filtrate to 1 to 10 mg / disc test sample and placing it in the center of the culture medium, and for 12 hours for bacteria in the incubator. In the case of a mold) was measured by incubation for 48 hours.

* 44 Minimum Inhibition Concentration Method (MIC) is defined as the minimum concentration of test fluid that can inhibit the growth of microorganisms. The test solution was added at various concentrations in the nutrient medium and batch cultured to measure MIC. Test samples were added to the nutrient medium and the nutrient mixture was sonicated for 5 minutes to prevent particle aggregation. Each mixture was then added to freshly prepared 1 ml of bacterial nutrient medium to maintain an initial bacterial concentration of 10 ^6-10 ⁷ CFUml ^-1 , then at 35 ° C. for 2 days for bacteria and 5 for fungus. Incubated in shaker at 25 ° C. for days. Gram-positive Staphylococcus aureus (ATCC 6538P), Gram-negative Escherichia coli (ATCC 8739), Pseudomonas aeruginosa (ATCC 9027), Candida albicans (ATCC 10231), and Aspergillus niger as filamentous fungi for the study of cosmetic antibacterial effects. (ATCC 22343) was used. Microbial growth of the strains was examined by observing at 650 nm with a microplate reader for 48 hours.

As shown in Table 1 and Table 2, Moringa oleifera extract extract using ethanol and ethyl acetate was Staphylococcus aureus (ATCC 6538P), Gram-negative Escherichia coli (ATCC 8739), Pseudomonas aeruginosa (ATCC 9027), Candida albicans (ATCC) 10231) and filamentous fungi showed significant antibacterial effects against Aspergillus niger (ATCC 22343). As a result of analyzing the extract, two bioactive substances, Niazimicin (FIG. 4) and 4- (α-L-rhamnopyranosyloxyl) -benzyl isothiocyanate (RBI) (FIG. 5), could be detected. The structure of the compound was measured by spectroscopic analysis by mass spectrophotometer, Infra red, 1D and 2D NMR.

Table 1

Antimicrobial Effects of Moringa Oleifera Seed Extract Using Ethanol (EtOH) According to Different Sample Concentrations for Various Microorganisms

microbe	Sample concentration% (w / v)
	0.005	0.01	0.05	0.1	0.5
Staphylococcus aureus	+	+	－	－	－
Escherichia coli	+	+	+	－	－
Pseudomonas aeruginosa	+	+	+	+	－
Candida albicans	+	+	+	+	－
Aspergillus niger	+	+	+	+	－

(+: Growth; Δ: growth inhibited compared to the control; −: inhibition)

TABLE 2

Antimicrobial Effect of Moringa Oleifera Extract Using Ethyl Acetate According to Different Sample Concentrations for Various Microorganisms

microbe	Sample concentration% (w / v)
	0.005	0.01	0.05	0.1	0.5
Staphylococcus aureus	+	－	－	－	－
Escherichia coli	+	+	+	－	－
Pseudomonas aeruginosa	+	+	+	+	－
Candida albicans	+	+	+	+	－
Aspergillus niger	+	+	+	+	－

(+: Growth; Δ: growth inhibited compared to the control;-: inhibition)

The two compounds showed antimicrobial activity against five microorganisms. In particular, RBI is known as Staphylococcus aureus (0.005%, w / v), Escherichia coli (0.1%, w / v), Pseudomonas aeruginosa (0.5%, w / v), Candida albicans (0.5%, w / v), Aspergillus It was shown to have strong antimicrobial activity against loose niger (0.5%, w / v) (Table 4). However, Niazimicin showed no antimicrobial activity except Staphylococcus aureus (0.1%, w / v).

The antimicrobial activity of RBI and Niazimicin was measured by the above-mentioned circular filtration method. Both RBI and Niazimicin showed antimicrobial activity when the test solution concentration was 5 mg / disc, but the growth inhibition region of RBI (indicating diameters of 23 mm and 28 mm, respectively) was wider than that of Niazimicin (FIG. 6).

The antimicrobial effect of the two compounds is assumed to be a molecular structure characteristic due to the molecular structure ethyl group. In other words, the structure of Niazimicin is not easy to penetrate into the microbial cell membrane because there is only one ethyl group in addition to glycoside, whereas the structure of RBI does not exist in the microbial cell membrane. It is shown that the growth inhibition mechanism can more easily penetrate the surface of the microbial cell membrane, while reducing the function of the cell membrane.

TABLE 3

Antimicrobial Effects of Niazimicin with Different Sample Concentrations on Various Microorganisms

microbe	Sample concentration% (w / v)
	0.005	0.01	0.05	0.1	0.5
Staphylococcus aureus	+	+	+	－	－
Escherichia coli	+	+	+	+	+
Pseudomonas aeruginosa	+	+	+	+	+
Candida albicans	+	+	+	+	+
Aspergillus niger	+	+	+	+	+

(+: Growth; Δ: growth inhibited compared to the control;-: inhibition)

Table 4

Antimicrobial Effects of 4- (α-L-rhamnopyranosyloxyl) -benzyl isothiocyanate (RBI) with Different Sample Concentrations on Various Microorganisms

microbe	Sample concentration% (w / v)
	0.005	0.01	0.05	0.1	0.5
Staphylococcus aureus	－	－	－	－	－
Escherichia coli	+	+	+	－	－
Pseudomonas aeruginosa	+	+	+	+	－
Candida albicans	+	+	+	+	－
Aspergillus niger	+	+	+	+	－

(+: Growth; Δ: growth inhibited compared to the control;-: inhibition)

The antimicrobial effect of porous zinc oxide was evaluated to examine the correlation between the properties of the porous material and the antimicrobial activity (Table 5). As a result, the antibacterial effect of the porous zinc oxide did not have antibacterial effect except Staphylococcus aureus and Escherichia coli.

Table 5

Antimicrobial Effects of Porous Zinc Oxide on Various Sample Concentrations for Various Microorganisms

microbe	Sample concentration% (w / v)
	0.004	0.01	0.05	0.1	0.5
Staphylococcus aureus	－	－	－	－	－
Escherichia coli	+	+	－	－	－
Pseudomonas aeruginosa	+	+	+	+	+
Candida albicans	+	+	+	+	+
Aspergillus niger	+	+	+	+	+

(+: Growth; Δ: growth inhibited compared to the control;-: inhibition)

Moringa oleifera extract and RBI (Table 8) using porous zinc oxide, an inorganic antimicrobial agent, as a support and ethanol (Table 6) and ethyl acetate (Table 7) were collected on the support to provide antimicrobial effects against various microorganisms. Evaluated. As a result, it was confirmed that the antimicrobial effect of Moringa oleifera seed extract and RBI was improved compared to the evaluation of the antimicrobial effect without a porous zinc oxide support. Since 10% of each natural substance was supported by zinc oxide, it can be seen that the antimicrobial effect was increased by about 10 to 50 times or more depending on the type of microorganism compared to before loading. That is, it was found that there is a synergistic antimicrobial effect between zinc oxide and Moringa oleifera seed extract. This is because, as mentioned earlier, the molecular structure of the ethyl-free RBI adhered zinc oxide to the microbial cell membrane more easily, thereby effectively inhibiting the growth of microorganisms by the mechanism by which Zn ⁺ reacts with the -SH group to inactivate enzymes. Shown.

Table 6

Antimicrobial Effect of Zinc Oxide Captured Ethanol (EtOH) Extract of Moringa oleifera Seed at Different Sample Concentrations for Various Microorganism

microbe	Sample concentration% (w / v)
	0.001	0.005	0.01	0.05	0.1	0.5
Staphylococcus aureus	+	+	－	－	－	－
Escherichia coli	+	+	+	－	－	－
Pseudomonas aeruginosa	+	+	+	+	+	－
Candida albicans	+	+	+	+	－	－
Aspergillus niger	+	+	+	+	－	－

(+: Growth; Δ: growth inhibited compared to the control;-: inhibition)

TABLE 7

Antibacterial Effect of Zinc Oxide from Moringa Oleifera Seed Ethyl Acetate Extract with Different Sample Concentrations for Various Microorganisms

microbe	Sample concentration% (w / v)
	0.001	0.005	0.01	0.05	0.1	0.5
Staphylococcus aureus	+	－	－	－	－	－
Escherichia coli	+	+	－	－	－	－
Pseudomonas aeruginosa	+	+	+	+	+	－
Candida albicans	+	+	+	+	+	－
Aspergillus niger	+	+	+	+	+	－

(+: Growth; Δ: growth inhibited compared to the control;-: inhibition)

Table 8

Antimicrobial Effects of Zinc Oxide Captured with 4- (α-L-rhamnopyranosyloxyl) -benzyl isothiocyanate (RBI) at Different Sample Concentrations on Various Microorganisms

microbe	Sample concentration% (w / v)
	0.001	0.005	0.01	0.05	0.1	0.5
Staphylococcus aureus	－	－	－	－	－	－
Escherichia coli	+	+	－	－	－	－
Pseudomonas aeruginosa	+	+	+	+	+	－
Candida albicans	+	+	+	+	－	－
Aspergillus niger	+	+	+	+	－	－

(+: Growth; Δ: growth inhibited compared to the control;-: inhibition)

Since the antimicrobial agent according to the present invention has excellent antimicrobial effect against microorganisms such as Staphylococcus aureus and Escherichia coli, and is excellent in safety, it is useful in cosmetics and the like.

Claims

An antimicrobial agent in which Moringa oleifera extract, 4- (α-L-rhamnopyranosyloxyl) -benzyl isothiocyanate (RBI) or Niazimicin is trapped in a porous material.
The antimicrobial agent of claim 1 wherein the porous material is zinc oxide (ZnO).
The antimicrobial agent of claim 1, wherein the Moringa oleifera extract is an Moringa oleifera seed extract.
The antimicrobial agent of claim 3, wherein the Moringa oleifera seed extract is Moringa oleifera ethanol extract or Moringa oleifera seed ethyl acetate extract.
The antimicrobial agent according to claim 1 or 3, wherein the Moringa oleifera extract contains 4- (α-L-rhamnopyranosyloxyl) -benzyl isothiocyanate (RBI) or Niazimicin as an active ingredient.
Moringa oleifera extract, 4- (α-L-rhamnopyranosyloxyl) -benzyl isothiocyanate (RBI) or Niazimicin to the porous material to produce an antimicrobial agent.
The method of claim 6, wherein the porous material is zinc oxide (ZnO).
The method of claim 6, wherein the Moringa oleifera extract is a Moringa oleifera seed extract.
The method of claim 8, wherein the Moringa oleifera seed extract is Moringa oleifera ethanol extract or Moringa oleifera seed ethyl acetate extract.
The method according to claim 8 or 10, wherein the Moringa oleifera extract comprises 4- (α-L-rhamnopyranosyloxyl) -benzyl isothiocyanate (RBI) or Niazimicin as an active ingredient.