KR20210042013A - Novel preservatives and preparation method thereof - Google Patents

Abstract

The present invention relates to a novel preservative and a method for manufacturing the same. The preservative of the present invention exhibits excellent antibacterial activity on gram-positive bacteria, gram-negative bacteria, yeast, and mold, and has high safety. In addition, the method for manufacturing the preservative of the present invention is a method for enzymatically converting a natural phenolic compound, which exhibits a high reaction conversion rate even under environmentally friendly solvent-free reaction conditions at room temperature and pressure in a short time with a small amount of enzyme.

Description

Novel preservatives and preparation method thereof {Novel preservatives and preparation method thereof}

The present invention relates to a novel preservative and a method of preparing the same.

Preservatives refer to raw materials/materials that play a role in inhibiting or reducing the growth of microorganisms during storage and use of the product. Preservatives are widely used not only during the shelf life of cosmetics, but also to prevent changes in physicochemically such as decay and deterioration due to contamination by microorganisms such as bacteria and fungi while consumers use the product. Preservatives are used not only in cosmetics, but also in many consumer goods such as food and medicine. Representative preservatives used in cosmetics include paraben and phenoxyethanol, but these preservatives are currently out of demand from consumers due to problems such as toxicity, skin irritation, and allergies. For this reason, in the current cosmetic market, products emphasizing that they do not contain preservatives are showing a strong trend, but in reality, these non-preservative cosmetics are mixed with a large amount of plant extracts that can act as an antiseptic substitute material or preservative. In the case of 1,2-hexanediol, which is used as a representative antiseptic substitute material in the cosmetic industry, it must be used at a high concentration to exhibit sufficient effects as a preservative, resulting in problems such as formulation, skin irritation, and feeling of use. Occurs. In addition, plant extracts are generally used in high concentration because their preservative effect is weak, causing problems such as discoloration and discoloration. Therefore, the development of new natural preservatives having excellent antiseptic effects is required.

Korean Patent Registration No. 1888233

An object of the present invention is to provide a novel preservative.

An object of the present invention is to provide a method for producing a novel preservative.

1. A preservative comprising a compound of Formula 1 below:

[Formula 1]

In Formula 1,

R ₁ is C1 to C6 alkyl;

R ₂ is hydrogen or C1 to C6 alkyl.

2. In the above 1, wherein R ₁ is C1 to C3 alkyl, and R ₂ is hydrogen or C1 to C3 alkyl, a preservative.

3. In the above 1, wherein Formula 1 is 4-hydroxy-3-methoxybenzyl propionate (4-hydroxy-3-methoxybenzyl propionate);

4-hydroxy-3-methoxybenzyl acetate;

4-hydroxy-3-methoxybenzyl butyrate;

3,4-dihydroxybenzyl propionate;

3,4-dihydroxybenzyl acetate; And

3,4-dihydroxybenzyl butyrate (3,4-dihydroxybenzyl butyrate) is at least one selected from the group consisting of, a preservative.

4. The preservative according to the above 1, wherein the preservative exhibits antimicrobial activity against at least one selected from the group consisting of Escherichia, Staphylococcus, Candida, and Aspergillus.

5. The preservative according to the above 1, wherein the preservative is at least one of a cosmetic preservative, a food preservative, and a pharmaceutical preservative.

6. A method for preparing a preservative comprising reacting a compound of the following formula 2 or a compound of the following formula 3, and a compound of the following formula 4 in the presence of a lipase:

[Formula 2]

(In Formula 2, R ₃ is C1 to C6 alkyl, and R ₆ is C1 to C6 alkyl)

[Formula 3]

(In Formula 3, R ₄ is C1 to C6 alkyl)

[Formula 4]

(In Formula 4, R ₅ is hydrogen or C1 to C6 alkyl).

7. In the above 6, wherein R ₃ is C1 to C3 alkyl, R ₄ is C1 to C3 alkyl, R ₅ is hydrogen or C1 to C3 alkyl, and R ₆ is C1 to C3 Alkyl phosphorus, a method for preparing a preservative.

8. In the above 6, wherein the reaction is carried out at 70 ℃ or less, the method for producing a preservative.

9. The method for preparing a preservative according to the above 6, wherein the reaction is carried out for a time period of 10 hours or less.

10. The method for preparing a preservative according to the above 6, wherein the compound of Formula 3 and the compound of Formula 4 are reacted in a 1: 1 to 3 molar ratio.

11. A cosmetic composition comprising the preservative of any one of the above 1 to 5.

12. The above 11, wherein the preservative is contained in an amount of 0.01 to 20% by weight based on the cosmetic composition, the cosmetic composition.

The preservative of the present invention exhibits excellent antibacterial activity against gram-positive bacteria, gram-negative bacteria, yeast, and mold.

The manufacturing method of the present invention is to enzymatically convert natural phenolic compounds obtained from biomass, which is a renewable resource, to prepare a preservative.With a small amount of enzyme, a high reaction conversion rate even under mild reaction conditions at room temperature and pressure within a short time. Represents.

1 shows the reaction scheme of the enzymatic esterification of vanillic acid and n-propanol.
Figure 2 shows the HPLC analysis results and reaction conversion of the enzymatic esterification reaction of vanillic acid and n-propanol.
3 shows the reaction scheme of the enzymatic transesterification reaction of vanillin alcohol and ethyl propionate.
4 shows the reaction conversion rate of the enzymatic transesterification reaction of vanillin alcohol and ethyl propionate according to the concentration of vanillin alcohol.
5 shows the reaction conversion rate of the enzymatic transesterification reaction of vanillin alcohol and ethyl propionate according to the concentration of the enzyme.
6 shows the reaction conversion rate of the enzymatic transesterification reaction of vanillin alcohol and ethyl propionate with or without molecular sieves treatment.
7 shows the results of analyzing an enzymatic transesterification reaction product of vanillin alcohol and ethyl propionate ^{through H 1 -NMR.}
8 shows the reaction scheme of the enzymatic transesterification reaction of vanillin alcohol and ethyl acetate.
9 shows the results of analyzing the reaction product of vanillin alcohol and ethyl acetate ^{through H 1 -NMR.}
10 is a comparison of the reaction conversion rates of the enzymatic transesterification of vanillin alcohol and ethyl acetate, the enzymatic transesterification of vanillin alcohol and ethyl propionate, and the enzymatic transesterification of vanillin alcohol and ethyl butyrate. Show one graph.
11 shows the reaction scheme of the enzymatic transesterification reaction of vanillin alcohol and ethyl butyrate.
12 shows the results of analyzing the reaction product of vanillin alcohol and ethyl butyrate ^{through H 1 -NMR.}
13 shows the results of confirming the antibacterial activity of vanillin propionate and vanillin butyrate by disc diffusion assay.
14 shows the results of confirming the antibacterial activity of vanillin propionate by MIC (Minimum Inhibitory Concentrations, mg/mL).
15 shows the results of evaluating the cytotoxicity of vanillin propionate through the WST-1 Assay (A: keratinocytes, B: dermal fibroblasts).
Fig. 16 shows the results of evaluating the cytotoxicity of vanillin propionate through L/D fluorescence staining (target cells: keratinocytes, green: living cells, red: dead cells).
Fig. 17 shows the results of evaluating the cytotoxicity of vanillin propionate through L/D fluorescence staining (target cells: dermal fibroblasts, green: living cells, red: dead cells).
18 shows the reaction scheme of the enzymatic transesterification reaction of vanillin alcohol and triacetin.
19 shows the reaction conversion rate of the enzymatic transesterification reaction of vanillin alcohol and triacetin.
Figure 20 shows the reaction scheme of the enzymatic transesterification reaction of 3,4-dihydroxybenzyl alcohol and ethyl propionate.
Figure 21 shows the reaction conversion rate of the enzymatic transesterification reaction of 3,4-dihydroxybenzyl alcohol and ethyl propionate.

The present invention provides a preservative comprising a compound of Formula 1:

[Formula 1]

R ₁ may be C1 to C6 alkyl. Specifically, R ₁ may be C1 to C3 alkyl.

R ₂ may be hydrogen or C1 to C6 alkyl. Specifically, R ₂ may be hydrogen or C1 to C3 alkyl.

Alkyl can be straight or branched chain alkyl.

The term “preservative” refers to a substance that is customarily added to a composition to preserve the composition from substances causing contamination.

The compound of Formula 1 is 4-hydroxy-3-methoxybenzyl propionate;

4-hydroxy-3-methoxybenzyl acetate;

4-hydroxy-3-methoxybenzyl butyrate;

3.4-dihydroxybenzyl propionate;

3,4-dihydroxybenzyl acetate; And

With 3,4-dihydroxybenzyl butyrate

It may be at least one selected from the group consisting of.

4-hydroxy-3-methoxybenzyl propionate; 4-hydroxy-3-methoxybenzyl acetate; 4-hydroxy-3-methoxybenzyl butyrate; 3,4-dihydroxybenzyl propionate; 3,4-dihydroxybenzyl acetate and 3,4-dihydroxybenzyl butyrate may exhibit antimicrobial and/or antibacterial and/or antifungal effects.

4-hydroxy-3-methoxybenzyl propionate; 4-hydroxy-3-methoxybenzyl acetate; 4-hydroxy-3-methoxybenzyl butyrate; 3,4-dihydroxybenzyl propionate; 3,4-dihydroxybenzyl acetate and 3,4-dihydroxybenzyl butyrate may each exhibit antibacterial effects against different bacteria.

4-hydroxy-3-methoxybenzyl propionate; 4-hydroxy-3-methoxybenzyl acetate; 4-hydroxy-3-methoxybenzyl butyrate; 3,4-dihydroxybenzyl propionate; A plurality of compounds among 3,4-dihydroxybenzyl acetate and 3,4-dihydroxybenzyl butyrate may be mixed and used as a preservative.

The preservative of the present invention may exhibit antibacterial activity against at least one of bacteria, fungi and yeast. The preservatives of the present invention can be used as antimicrobial and/or antibacterial and/or antifungal agents.

Bacteria may be any of Enterococcus (Enterococcus) genus, the Escherichia (Escherichia) genus, Staphylococcus (Staphylococcus) genus Pseudomonas (Pseudomonas) in and Burke holde Liao at least one of the inside (Burkholderia), but is not limited thereto. For example, bacteria are Enterococcus faecalis , Escherichia coli , Staphylococcus aureus , Pseudomonas aeruginosa , Burkholderia sepacia ( Burkholderia cepacia ).

The fungus may be of the genus Aspergillus , but is not limited thereto. For example, the fungus may be Aspergillus niger.

Yeast may be of the genus Candida , but is not limited thereto. For example, the yeast may be Candida albicans.

The preservative of the present invention may be at least one of a cosmetic preservative, a food preservative, and a pharmaceutical preservative.

The preservative of the present invention may be included in a cosmetic composition, a pharmaceutical composition, or a food composition to exhibit a preservative power capable of preserving the composition from contamination by microorganisms.

The term "preservation power" refers to the defense power that can prevent the product from deteriorating and maintain its original state.

The preservative of the present invention may not impair the texture, color, or odor of a cosmetic composition, pharmaceutical composition or food composition containing the preservative.

The preservative of the present invention may not contain known chemical preservatives other than the compound of Formula 1 above.

The preservative of the present invention may not contain chemical preservatives such as parabens and phenoxyethanol, and in that case, problems such as toxicity, skin irritation, and allergy caused by the use of chemical preservatives can be solved. In addition, the preservative of the present invention may exhibit the preservative power or antibacterial power similar to or superior to the preservative power or antibacterial power of the conventional chemical preservatives of parabens.

In addition, the present invention provides a method for preparing a preservative.

The method for preparing a preservative of the present invention may include reacting a compound of Formula 2 or Formula 3 and a compound of Formula 4 in the presence of a lipase (hereinafter, a substrate reaction step).

[Formula 2]

In Formula 2, R ₃ may be C1 to C6 alkyl. Specifically, R ₃ may be C1 to C3 alkyl.

In Formula 2, R ₆ may be C1 to C6 alkyl. Specifically, R ₆ may be C1 to C3 alkyl.

[Formula 3]

In Formula 3, R ₄ may be C1 to C6 alkyl. Specifically, R ₄ may be C1 to C3 alkyl.

[Formula 4]

In Formula 4, R ₅ may be hydrogen or C1 to C6 alkyl. Specifically, R ₅ may be C1 to C3 alkyl.

The compound of Formula 4 may be a phenolic alcohol. For example, the compound of Formula 4 may be vanillin alcohol or 3,4-dihydroxybenzyl alcohol.

Vanillin alcohol or 3,4-dihydroxybenzyl alcohol may be naturally obtained or synthesized. Vanillin alcohol may be produced through a reduction reaction of vanillin, a natural phenolic compound.

In the substrate reaction step, the compound of Formula 2 and the compound of Formula 3 are used as an acyl donor, and the compound of Formula 4 is used as an acyl acceptor.

When the compound of Formula 3 is used as an acyl donor in the substrate reaction step, glycerol is produced as a by-product.

Since glycerol is a representative material used as a moisturizing component of cosmetics, when the preservative prepared by the method of the present invention is used as a preservative for cosmetics, it is economical because a separate separation process for removing by-products from the material produced after the reaction is not required.

The substrate reaction step may be performed under no solvent. This is because a separate organic solvent is not required since the compound of Formula 2 or the compound of Formula 3 is a liquid substrate.

The lipase used in the substrate reaction step can be used without limitation, known in the art. For example, the lipase may be Candida antarctica lipase (CAL), Rhizomucor miehei lipase (RML), Rhizopus javanicus lipase (RJL), Rhizopus delemar lipase (RDL). , Aspergillus niger lipase (ANL), Aspergillus oryzae lipase (AOL), Candida cylindracea lipase (CCL), Pseudomonas fluorescence lipase (PFL), porcine pancreatic lipase (PPL) and immobilized forms thereof (e.g. Novozym 435 ^TM ) It may be at least one selected from the group consisting of.

The concentration of lipase used in the substrate reaction step may be 50 U/mL or less, for example, 50 U/mL or less, 45 U/mL or less, 40 U/mL or less, 35 U/mL or less, 30 U/mL or less, It may be 25 U/mL or less, 20 U/mL or less, 15 U/mL or less, 10 U/mL or less, or 5 U/mL or less. The lower limit of the lipase concentration used in the substrate reaction step is 0.1 U/mL, 0.5 U/mL, 1 U/mL, 1.5 U/mL, 2 U/mL, 2.5 U/mL, 3 U/mL, 3.5 U/mL. , 4 U/mL or 4.5 U/mL.

When the compound of Formula 4 is used as a reactant instead of vanillic acid in the substrate reaction step, reaction conversion is high even in the presence of lipase at a concentration of 50 U/mL or less. On the other hand, in the case of obtaining the same reaction product using vanillic acid other than the compound of Formula 4 as a reactant, the reaction conversion rate is not high even if the reaction is carried out in the presence of a lipase of about 8 times higher than the above concentration.

The substrate reaction step may be performed under a temperature condition of 70° C. or less. For example, the substrate reaction step is 70°C or less, 65°C or less, 60°C or less, 55°C or less, 50°C or less, 45°C or less, 40°C or less, 35°C or less, 30°C or less, 25°C or less, 20°C or less , It may be performed under a temperature condition of 15°C or less, 10°C or less, or 5°C or less. The lower limit of the temperature condition of the substrate reaction step may be 5°C, 10°C, 15°C, 20°C, 25°C, 30°C or 35°C.

Although the reaction temperature conditions may vary depending on the type or concentration of the substrate used in the substrate reaction step, the reaction conversion rate is high even at a low temperature condition of 70° C. or less when the compound of Formula 4 is used as a reactant. On the other hand, in the case of obtaining the same reaction product using vanillic acid, not the compound of Formula 4 as a reactant, the reaction conversion rate is not high even if the reaction is carried out under a high temperature condition exceeding 70°C.

In the substrate reaction step, the reaction may be performed for a period of 10 hours or less. For example, the substrate reaction step is performed for a time period of 10 hours or less, 9 hours or less, 8 hours or less, 7 hours or less, 6 hours or less, 5 hours or less, 4 hours or less, 3 hours or less, 2 hours or less, or 1 hour or less. It can be. The lower limit of the reaction execution time in the substrate reaction step may be 30 minutes, 1 hour, or 1 hour 30 minutes.

When the compound of Formula 4 is used instead of vanillic acid in the substrate reaction step, the reaction conversion rate is high even if the reaction is performed for a short time of 10 hours or less. On the other hand, in the case of obtaining the same reaction product using vanillic acid other than the compound of Formula 4 as a reactant, the reaction conversion rate is not high even if the reaction is performed for more than 10 hours.

The concentration of the compound of Formula 4 used in the substrate reaction step may be 10 to 300 mM, for example, the concentration of vanillin alcohol is 10 to 300 mM, 20 to 290 mM, 30 to 280 mM, 40 to 270 mM, 50 to 260 mM, 60 to 250 mM, 70 to 240 mM, 80 to 230 mM, 90 to 220 mM, 100 to 210 mM, 110 to 200 mM, 120 to 190 mM, 130 to 180 mM, 140 to 170 mM, 150 to It may be 160 mM, but is not limited thereto.

The substrate reaction step may be to react the compound of Formula 2 and the compound of Formula 4 in a molar ratio of 1 to 200:1. For example, the substrate reaction step may be to react the compound of Formula 2 and the compound of Formula 4 in a molar ratio of 1 to 200:1, 10 to 100:1, 20 to 50:1, 30 to 50:1, Specifically, it may be reacted at a molar ratio of 30 to 50:1.

The substrate reaction step may be to react the compound of Formula 3 and the compound of Formula 4 in a 1: 1 to 3 molar ratio. For example, the substrate reaction step may be to react the compound of Formula 3 and the compound of Formula 4 in a 1: 1 to 3 molar ratio or 1: 1.5 to 2.5 molar ratio.

Additionally, the present invention can provide a cosmetic composition comprising the preservative described above.

The cosmetic composition of the present invention exhibits excellent preservation power and safety by including the preservative described above.

The content of the preservative contained in the cosmetic composition of the present invention is not particularly limited as long as the desired preservative power can be secured, and those skilled in the art can determine the appropriate content according to the degree of desired preservative power.

The preservative of the present invention may be included in an amount of 0.01 to 20% by weight based on the total cosmetic composition. For example, the preservative of the present invention may be included in an amount of 0.01 to 20% by weight, 0.1 to 10% by weight, 0.1 to 5% by weight, or 0.1 to 2% by weight based on the total cosmetic composition.

The cosmetic composition may be any known formulation, for example, skin, emulsion, cream, sun cream, foundation, essence, gel, pack, mask pack, foam cleanser, body cleanser, softening lotion, eyeliner, shampoo, conditioner, soap , Hairdressing agent, wool agent, hair cream, hair styling gel, lubricant, toothpaste, and may be one formulation selected from the group consisting of wet tissues, but is not limited thereto.

The cosmetic composition of the present invention may further contain known additives within a range that does not impair the effect of the preservative of the present invention depending on the formulation. For example, the cosmetic composition of the present invention further comprises an additive selected from the group consisting of a carrier, an emulsifier, a moisturizer, a skin conditioning agent, a surfactant, a chelating agent, an antioxidant, a fungicide, a stabilizer, and any combination thereof. I can.

Carrier is animal fiber, plant fiber, wax, paraffin, starch, tragacanth, cellulose derivative, polyethylene glycol, silicone, bentonite, silica, talc, zinc oxide, lactose, silica, aluminum hydroxide, calcium silicate, polyamide powder , Water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butyl glycol oil, glycerol aliphatic ester, polyethylene glycol, liquid diluent, ethoxylated isostearyl alcohol, polyoxy Ethylene sorbitol ester, polyoxyethylene sorbitan ester, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar or tragacanth, aliphatic alcohol sulfate, aliphatic alcohol ether sulfate, sulfosuccinic acid monoester, isethionate, imidazoli In the group consisting of nium derivatives, methyltaurates, sarcosinates, fatty acid amide ether sulfates, alkylamidobetaines, fatty alcohols, fatty acid glycerides, fatty acid diethanolamides, vegetable oils, linoline derivatives and ethoxylated glycerol fatty acid esters It may be at least one selected, but is not limited thereto.

The emulsifier may be at least one selected from the group consisting of liquid paraffin, cetyloctanoate, and stearic acid, but is not limited thereto.

The moisturizing agent may be at least one selected from the group consisting of glycerin and glyceryl stearate, but is not limited thereto.

The skin conditioning agent may be at least one selected from the group consisting of saliclomethicone and dimethicone, but is not limited thereto.

The surfactant may be at least one selected from the group consisting of cetostearyl alcohol, triethanolamine, and carboxyvinyl polymer, but is not limited thereto.

The chelating agent may be at least one selected from the group consisting of sodium ethylenediaminetetraacetate (EDTA), α-hydroxy fatty acid, lactoferrin, α-hydroxy acid, citric acid, lactic acid, malic acid, bilirubin, and biliverdin. However, it is not limited thereto.

The antioxidant may be at least one selected from the group consisting of butylhydroxyanisole, dibutylhydroxytoluene, and propyl gallate, but is not limited thereto.

In addition, the cosmetic composition of the present invention may further include at least one of a fat or oil component, an emollient agent, an organic pigment, an inorganic pigment, an organic powder, an ultraviolet absorber, a pH adjuster, an alcohol, a colorant, a fragrance, a blood circulation accelerator, a cooling agent, and a limiting agent. However, it is not limited thereto.

Hereinafter, examples will be described in detail to illustrate the present invention in detail.

Comparative Example: Preparation of a preservative using vanillic acid

In order to synthesize a compound having a structure similar to that of paraben, an enzymatic esterification reaction of vanillic acid, a natural phenolic compound, was performed. Vanillic acid was used as an acyl donor, and n-propanol was used as an acyl acceptor. The reaction was carried out in a solvent-free system by replacing the solvent by using an excess of the liquid n-propanol substrate (see Fig. 1). Novozym 435, the most representative lipase, was used as the lipase enzyme.

Specifically, the esterification reaction between vanillic acid and n-propanol is a solvent-free system based on a concentration of vanillic acid of 83 mM (molar ratio; vanillic acid: n-propanol = 1: 160), a total volume of 2.4 mL, molecular sieves 20 mg/ mL, enzyme concentration of 420 U/mL, 70 °C, and reacted for at least 10 hours in a multi-hot plate at 300 rpm. At this time, fractionated by reaction time, diluted 20 times, and analyzed by HPLC under 275 nm conditions (see FIG. 2). As a result, it showed a very low reaction conversion rate (conversion) within 5% of the case.

Example 1: Preparation of a preservative using vanillin alcohol and short chain ester

1. Manufacturing method

Instead of the esterification reaction between vanillic acid and n-propanol, which has a very low conversion rate (Comparative Example 1), vanillin alcohol, a natural phenolic compound, is used as an acyl acceptor substrate, and a short chain ester is used as an acyl donor ( acyl donor) was used as a substrate for enzymatic transesterification.

The short-chain ester includes ethyl acetate, ethyl propionate, and ethyl butyrate used in the following reaction, and is a short-chain hydrocarbon (for example, in the ester group (-COO-)). It refers to a compound to which a chain having C1-6 carbon atoms) is bonded.

1-1. Preparation of 4-hydroxy-3-methoxybenzyl propionate

An enzymatic transesterification reaction was performed using vanillin alcohol, a natural phenolic compound, as an acyl acceptor substrate and ethyl propionate as an acyl donor substrate. At this time, the reaction was carried out in a solvent-free system by replacing the solvent with ethyl propionate as a liquid (see FIG. 3).

Specifically, liquid ethyl propionate (total volume 2 mL) and solid vanillin alcohol (50, 100, 200 mM) were prepared and completely dissolved in a 20 mL glass vial. The molar ratio of ethyl propionate and 50mM vanillin alcohol was 173:1, the molar ratio of ethyl propionate and 100mM vanillin alcohol was 87:1, and the molar ratio of ethyl propionate and 200mM vanillin alcohol was 43:1.

In order to remove ethanol, which is produced as a by-product of the transesterification reaction between vanillin alcohol and ethyl propionate, 20 mg/mL of molecular sieves (4Å) was added to the above solution and prepared together. Novozym 435 (5, 10, 20 U/mL) was added to the prepared solution and the reaction was initiated. The reaction solution was reacted in a shaking incubator at 35 °C to 40 °C and 200 rpm for up to 5 hours.

The performance of the transesterification reaction was confirmed by analysis by High Performance Liquid Chromatography (HPLC). Depending on the reaction time, 10 μL was aliquoted from the stock solution, diluted 30 times with HPLC grade methanol, and subjected to HPLC analysis. Analysis was performed by HITACHI's Primaide PM 1000 Series HPLC, and TSKgel® ODS-100Z C18 Column (4.6 mm × 15 cm, 5 μm particle size; Tosoh Bioscience GmbH, Stuttgart, Germany) was used for separation. The elution conditions were prepared with solvent A: methanol 100%, solvent B: acetic acid 0.1% (with HPLC grade water), and solvent C: water 100%, and analyzed by setting a flow rate of 1 mL/min. The elution time conditions are as follows; 0-2 min: A 50%-B 50 %, 2-7 min: A 90%-B 10 %, 7-10 min: A 90%-B 10 %, 10-15 min: A 50%-B 50 %. All samples were detected by Diode Array Detector (DAD) at 280 nm.

Enzyme concentration 10 U/mL, molecular sieves 20 mg/mL, 35 °C, 200 rpm.Reaction conversion rate regardless of the concentration of vanillin alcohol even if the reaction is carried out at different concentrations of 50, 100, and 200 mM vanillin alcohol. Was found to be more than 90% (see Fig. 4).

Vanillin alcohol concentration 100 mM, molecular sieves 20 mg/mL, 35 °C, 200 rpm, and even if the enzyme concentration is changed to 5, 10, 20 U/mL, the reaction conversion rate is 90 regardless of the enzyme concentration. % Or more (see Fig. 5).

As shown in FIGS. 4 and 5, the conversion rate could be greatly improved by using vanillin alcohol instead of vanillic acid as a reaction substrate.

Additionally, at the concentration of vanillin alcohol 100 mM and 200 mM, the remaining conditions are the same, and whether or not 20 mg/mL of molecular sieves (4Å) is treated (W/ms: molecular sieves treatment, W/o ms: molecular sieves no treatment) ), the conversion rate after the reaction for 3 hours was compared. As a result, it was found that treatment with molecular sieves results in a higher conversion rate (see FIG. 6).

After the reaction was completed, the reaction stock solution was transferred to a 15 mL centrifuge tube, and then molecular sieves (4Å) and enzyme were separated and removed through centrifugation at 10,000 rpm for 10 to 15 minutes. Evaporation was performed for 30-40 minutes in a vacuum rotary evaporator at 60°C to remove the liquid ethyl propionate remaining without reaction and to proceed with concentration. The recovered product was stored in a vacuum desiccator for 2 to 3 days, and then ethyl propionate was completely removed. The chemical structure of the finally obtained material was ^{analyzed through 400 MHz H 1} -NMR, and the analysis result was consistent with the chemical structure of 4-hydroxy-3-methoxybenzyl propionate (vanillin propionate) (see FIG. 7 ). ).

1-2. Preparation of 4-hydroxy-3-methoxybenzyl acetate

Instead of ethyl propionate as an acyl donor, ethyl acetate with one less carbon number was used, and the rest were reacted under the same conditions as in Example 1 1-1.

The enzymatic transesterification reaction was carried out for 5 hours under the conditions of ethyl acetate with vanillin alcohol 100 mM, enzyme 10 U/mL, molecular sieves 20 mg/mL, 35 °C, 200 rpm (see Fig.8: vanillin alcohol and Enzymatic transesterification reaction with ethyl acetate as a reactant). As a reaction product, 4-hydroxy-3-methoxybenzyl acetate (vanillin acetate) was produced. The above-described reaction between ethyl acetate and vanillin alcohol showed a high reaction conversion rate of 90% or more (see FIG. 10).

After the reaction was completed, the reaction stock solution was transferred to a 15 mL centrifuge tube, and then molecular sieves (4Å) and enzyme were separated and removed through centrifugation at 10,000 rpm for 10 to 15 minutes. Evaporation was performed for 30 to 40 minutes in a vacuum rotary evaporator at 50 °C to remove the remaining liquid ethyl acetate without reaction and to proceed with concentration. The recovered product was completely dried after storage for 2 to 3 days in a vacuum desiccator. As a result of analyzing the chemical structure of the finally obtained material ^{through 400 MHz H 1} -NMR, it was consistent with the chemical structure of 4-hydroxy-3-methoxybenzyl acetate (see FIG. 9).

1-3. Preparation of 4-hydroxy-3-methoxybenzyl butyrate

Instead of ethyl propionate as an acyl donor, ethyl butyrate with one more carbon number was used, and the rest were reacted under the same conditions as in Example 1 1-1.

Specifically, an enzymatic transesterification reaction was performed for 5 hours under the conditions of ethyl butyrate with vanillin alcohol 100 mM, enzyme 10 U/mL, molecular sieves 20 mg/mL, 35 °C, 200 rpm (see FIG. 11). Enzymatic transesterification reaction with vanillin alcohol and ethyl butyrate as reactants). As a result, 4-hydroxy-3-methoxybenzyl butyrate (vanillin butyrate) was produced. The above-described reaction between ethyl butyrate and vanillin alcohol showed a high reaction conversion rate of 90% or more (see FIG. 10). As a result of analyzing the chemical structure of the finally obtained material ^{through 400 MHz H 1} -NMR, it was consistent with the chemical structure of 4-hydroxy-3-methoxybenzyl butyrate (see FIG. 12). As a result, regardless of the type of short-chain ester used in the reaction, the reaction between the short-chain ester and vanillin alcohol all showed a high reaction conversion rate of 90% or more (see FIG. 10).

2. Evaluation of antibacterial efficacy

2-1. Evaluation of antibacterial efficacy through paper disc diffusion assay

A paper disc diffusion assay was performed to determine the antimicrobial efficacy of the vanillin ester (vanillin propionate, vanillin acetate, and vanillin butyrate) prepared by the above method. Microorganisms used to evaluate antibacterial efficacy are shown in Table 1 below.

The microorganisms in Table 1 are pathogenic microorganisms, and are representative microorganisms used when evaluating the efficacy of cosmetic preservative components.

To proceed with the paper disc diffusion assay, a disc obtained by soaking a certain amount (20 μL) of a sample was placed on a plate coated with pathogenic bacteria and cultured. When the treated sample has an antibacterial effect, a clear zone is formed because the treated sample spreads to a disc (diameter: 8 mm) and inhibits the growth of bacteria. This transparent ring is referred to as the microbial growth inhibition zone and is also referred to as the inhibition zone. The antibacterial effect is evaluated by measuring the diameter (unit: mm) of the transparent ring created on the disc, and it can be interpreted that the larger the diameter of the inhibition zone is, the better the antibacterial effect is (see FIG. 13(A)).

Since the ingredients that can be used as preservatives can be used up to 2 wt% as a single ingredient for the entire cosmetics, the antibacterial efficacy was evaluated at 2 wt% for all samples. DMSO was used as a solvent to make a 2 wt% sample stock solution.

As a sample, a vanillin ester such as vanillin propionate and vanillin butyrate prepared by the 1. Preparation method of Example 1 was used, and as a control, the existing preservatives methyl paraben, ethyl paraben, propyl paraben, butyl paraben, phenoxyethanol , 1,2-hexanediol and vanillin alcohol as a substrate material before the reaction were used.

As a result of antibacterial efficacy evaluation, vanillin ester showed antibacterial efficacy, and in particular, vanillin butyrate showed excellent antibacterial efficacy comparable to butyl paraben in all of E. coli , S. aureus and C. albicans strains (see FIG. 13(B)). , ND: not detected). If various kinds of vanillin ester are used together as a preservative, it can be expected to produce synergistic effects.

2-2. Evaluation of antibacterial efficacy by checking Minimum Inhibitory concentration (MIC)

Additional antibacterial efficacy evaluation was performed by checking Minimum Inhibitory Concentration (MIC). MIC is the minimum inhibitory concentration, and the smaller this value, the better the antimicrobial efficacy of the sample.

The MIC of the vanillin propionate prepared by the above method was confirmed, and compared with the MIC values of the existing preservatives (methyl paraben, ethyl paraben, propyl paraben, phenoxyethanol, 1,2-hexanediol). As a result of checking the MIC value, the vanillin ester showed significantly lower MIC values compared to phenoxyethanol and 1,2-hexanediol. In addition, it exhibited similar or lower MIC values compared to the parabens compound (see FIG. 14).

For reference, such as methyl paraben, ethyl paraben, and propyl paraben of FIG. 13(B), the types of bacteria and antibacterial activity may vary according to the carbon length of the substituent. Thus, mixing vanillin acetate, vanillin propionate, and vanillin butyrate obtained using various types of acyl donor substrates is expected to exhibit more excellent antibacterial efficacy against various bacteria.

3. Cytotoxicity assessment

Cytotoxicity evaluation was performed on the vanillin ester and the positive control parabens. Cytotoxicity evaluation was performed on human keratinocytes (HaCaT cells) and human dermal fibroblasts (HDFs), which are cells that make up the skin.If toxicity to skin cells is low, it is possible to commercialize it in the cosmetic industry. It becomes higher. Vanillin propionate; Propyl parabens similar in structure to this; Ethyl paraben, which has similar antimicrobial efficacy, was used as a sample for toxicity evaluation, and WST-1 Assay and L/D fluorescence staining were performed as a toxicity evaluation method.

3-1. WST-1 Assay

The metabolic activity of mitochondria in cells was quantitatively measured through WST-1 Assay.

As a result of testing on keratinocytes, ethyl paraben and propyl paraben did not show cytotoxicity up to 0.5 mM, whereas vanillin propionate did not show cytotoxicity up to a higher concentration of 1.5 mM. In addition, propyl parabens all killed cells at a concentration of 2.5 mM, ethyl parabens showed a cell survival rate of about 40.5%, and vanillin propionate showed a cell survival rate of about 54.1% higher than this (Fig. 15 ( A) see). Similar results were also shown in the test for dermal fibroblasts (see Fig. 15(B)).

3-2. L/D fluorescence staining

In addition, vanillin propionate through L/D fluorescence staining of keratinocytes and dermal fibroblasts; Propyl paraben; The cytotoxicity of ethyl paraben was measured. Live cells are stained green, and dead cells are stained red.

Fig. 16 shows the results of L/D fluorescence staining of keratinocytes, and Fig. 17 shows the results of L/D fluorescence staining of dermal fibroblasts. It can be seen that the results of L/D fluorescence staining in FIGS. 16 and 17 also show the same trend as the results of the WST-1 assay.

Example 2: Preparation of a preservative using vanillin alcohol and triacetin

1. Manufacturing method

4-hydroxy-3-methoxybenzyl acetate (vanillin acetate) prepared in 1-2 of Example 1 was synthesized using another substrate. Triacetin was used instead of ethyl acetate as an acyl donor. Like 1-2 in Example 1, the reaction was carried out in an eco-friendly solvent-free system by replacing the solvent by using an excess amount of triacetin, which is a liquid (see FIG. 18: Enzymatic transester using vanillin alcohol and triacetin as reactants. Fire reaction). Through the above reaction, the same vanillin acetate product as the product of Example 1 1-2 may be obtained. Hereinafter, a product containing vanillin acetate obtained by reacting vanillin alcohol and triacetin is expressed as a triacetin product.

Triacetin, which is used as an acyl donor in the above reaction, is a known GRAS (Generally Recognized As Safe) substance and is widely used as a food additive, and can be used as it is without removing the remaining triacetin after the reaction is completed. In addition, glycerol, which is produced as a by-product of the above reaction, is a representative cosmetic moisturizing component, so there is no need to remove it after the reaction.

However, compared with the vanillin acetate obtained by the 1-2 method of Example 1, the purity of vanillin acetate is low because triacetin, a substrate added in excess to the material obtained after the reaction, and glycerol, a by-product of the reaction, are mixed together. Nevertheless, the manufacturing method of Example 2 has the advantage that a more economical process can be provided by eliminating the separation process that requires considerable energy and cost.

2. Evaluation of reaction conversion rate

The reaction conversion rate for the enzymatic synthesis reaction presented in the above 1 was confirmed. As described above, molecular sieves (4Å) were not used during the reaction because glycerol, which is produced as a reaction by-product, does not need to be removed.

100 mM vanillin alcohol and triacetin solution, a total volume of 2 mL, an enzyme concentration of 10 U/mL, 40 °C, and the result of the reaction for 5 hours under the conditions of 20 rpm are shown in FIG. 19(A). In addition, in order to prepare a triacetin product containing the highest concentration of vanillin acetate, a 1 M concentration of vanillin alcohol and triacetin solution, a total volume of 2 mL, an enzyme concentration of 20 U/mL, 70 °C, 400 rpm for 6 hours. The results of the reaction are shown in Fig. 19(B).

3. Synthesis of Triacetin Product Containing High Concentration Vanillin Ester

As described above, since the triacetin product contains by-products, antibacterial efficacy may be lower than that of the same amount of the preservative obtained by the synthesis method of Example 1 1-2. Accordingly, various triacetin products were synthesized by adjusting the molar ratio of the reactants so that the triacetin product may contain a high concentration of vanillin acetate. The reaction conditions were reacted for 4 hours at 60° C. and 200 rpm in a shaking incubator, and then biosynthesis was terminated. Table 2 shows the molar concentration of vanillin alcohol, the molar ratio of the reactant, the amount of enzyme used in the reaction, the reaction conversion rate, and the purity.

Concentration (M) Molar ratio Enzyme amount (U) Conversion (%) Purity (%) Vanillyl alcohol Triacetin 5.3 One
(4 mmol) One
(4 mmol) 800 90 47 10.7 2
(4 mmol) One
(2 mmol) 800 75 56 16 3
(6 mmol) One
(2 mmol) 1200 65 56

4. Confirmation of antibacterial efficacy of triacetin product

In Table 2, the antimicrobial efficacy was evaluated using a triacetin product (vanillyl alcohol: triacetin = 2: 1 reaction product) obtained by reacting with a vanillin alcohol concentration of 10.7 M in consideration of purity and reaction conversion rate and triacetin as a pre-reaction material. Antibacterial efficacy was evaluated in the same manner as in the 2-1 paper disc diffusion assay of Example 1, and the liquid sample was evaluated based on 2 wt%.

Despite the low purity of vanillin acetate, it was confirmed that the triacetin product exhibited antibacterial efficacy against E. coli and C. albicans (see Table 3).

Example 3: Preparation of a preservative using 3,4-dihydroxybenzyl short chain ester

1. Manufacturing method

In place of the vanillin alcohol used in Example 1, 3,4-dihydroxybenzyl alcohol was used as an acyl receiver to perform a transesterification reaction. 3,4-dihydroxybenzyl alcohol was used as an acyl acceptor, and ethyl propionate was used as an acyl donor. The reaction was carried out in a solvent-free system by replacing the reaction solvent using an excess of liquid ethyl propionate (see Fig. 20: Enzymatic trans using 3,4-dihydroxybenzyl alcohol and ethyl propionate as reactants. Esterification reaction). As a reaction product, 3,4-dihydroxybenzyl propionate was produced.

Specifically, 3,4-dihydroxybenzyl alcohol 100 mM and ethyl propionate solution, total volume 2 mL, enzyme concentration 20 U/mL, molecular sieves 20 mg/mL, 40 °C, 200 rpm under conditions of 5 The enzymatic transesterification reaction proceeded for a period of time. After 1 hour of reaction, a high reaction conversion rate of 90% or more was exhibited (see FIG. 21). At this time, the molar ratio of ethyl propionate and 3,4-dihydroxybenzyl alcohol was reacted at 87:1.

2. Evaluation of antibacterial efficacy

After the end of the final reaction time, the recovered product 3,4-dihydroxybenzyl propionate (purity 80%) was used to evaluate the antibacterial efficacy. Antibacterial efficacy was evaluated in the same manner as in the 2-1 paper disc diffusion assay of Example 1, and the liquid sample was evaluated based on 2 wt%. As a result, 3,4-dihydroxybenzyl propionate showed antibacterial efficacy against all strains of E. coli , S. aureus and C. albicans , showing a broader antibacterial spectrum than vanillin propionate (see Table 4). ).

Through the above examples, the enzymatic transesterification reaction of phenolic alcohols (vanillin alcohol, 3,4-dihydroxybenzyl alcohol) and short chain esters (ethyl acetate, ethyl propionate, ethyl butyrate) under solvent-free conditions. As a result, it was confirmed that a compound that can be used as a preservative can be prepared with high efficiency.

In addition, the compounds prepared by the transesterification reaction exhibit lower toxicity than synthetic substances (eg, parabens) used as preservatives, and at the same time, E. coli , S. aureus , C. albicans and A. niger It was confirmed that the antibacterial effect was similar to or better than the existing preservatives against various bacteria such as. Therefore, the compound prepared by the transesterification reaction may be used as a new preservative to replace the existing synthetic material.

Claims (12)

Preservatives comprising a compound of Formula 1:
[Formula 1]

In Formula 1,
R ₁ is C1 to C6 alkyl;
R ₂ is hydrogen or C1 to C6 alkyl.
The preservative of claim 1, wherein R ₁ is C1 to C3 alkyl, and R ₂ is hydrogen or C1 to C3 alkyl.
The method according to claim 1, wherein the formula 1 is 4-hydroxy-3-methoxybenzyl propionate (4-hydroxy-3-methoxybenzyl propionate);
4-hydroxy-3-methoxybenzyl acetate;
4-hydroxy-3-methoxybenzyl butyrate;
3,4-dihydroxybenzyl propionate;
3,4-dihydroxybenzyl acetate; And
3,4-dihydroxybenzyl butyrate (3,4-dihydroxybenzyl butyrate) is at least one selected from the group consisting of, a preservative.
The method according to claim 1, wherein the preservative is Escherichia (Escherichia) genus, Staphylococcus (Staphylococcus) genus Candida (Candida) genus and Aspergillus will represent an antimicrobial with respect to at least one selected from the group consisting of genus (Aspergillus) in Phosphorus, a preservative.
The preservative of claim 1, wherein the preservative is at least one of a cosmetic preservative, a food preservative, and a pharmaceutical preservative.
A method for preparing a preservative comprising reacting a compound of Formula 2 or Formula 3 and a compound of Formula 4 in the presence of a lipase:
[Formula 2]

(In Formula 2, R ₃ is C1 to C6 alkyl, and R ₆ is C1 to C6 alkyl)
[Formula 3]

(In Formula 3, R ₄ is C1 to C6 alkyl)
[Formula 4]

(In Formula 4, R ₅ is hydrogen or C1 to C6 alkyl).
The method according to claim 6, wherein R ₃ is C1 to C3 alkyl, R ₄ is C1 to C3 alkyl, R ₅ is hydrogen or C1 to C3 alkyl, and R ₆ is C1 to C3 alkyl. , Preservative manufacturing method.
The method of claim 6, wherein the reaction is carried out at 70°C or less.
The method of claim 6, wherein the reaction is carried out for a time period of 10 hours or less.
The method of claim 6, wherein the compound of Formula 3 and the compound of Formula 4 are reacted in a molar ratio of 1: 1 to 3.
A cosmetic composition comprising the preservative of any one of claims 1 to 5.
The cosmetic composition of claim 11, wherein the preservative is contained in an amount of 0.01 to 20% by weight based on the cosmetic composition.

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