KR20130052386A - Bacterial synthesized citrus cellolose for wound healing and dressing materials containing the same - Google Patents

Abstract

PURPOSE: A citrus-derived bacteria cellulose gel for wound healing and a dressing containing the same are provided to reduce initial inflammation reaction, to promote contraction through accumulation of extracellular matrix, and to enhance wound healing effects. CONSTITUTION: A cellulose gel for wound healing is prepared by extracting citrus using Gluconacetobacter. A dressing for wound healing contains the cellulose gel as an active ingredient. The dressing is a hydrocolloid or wet foam type. The dressing reduces initial inflammation reaction, promotes contraction through accumulation of extracellular matrix, and has wound healing effects. [Reference numerals] (AA) Wound area(%); (BB) Elapsed days after procedure(days)

Description

Citrus-derived bacterial cellulose gel for wound healing and dressing agent containing the same {Bacterial synthesized citrus cellolose for wound healing and dressing materials containing the same}

The present invention relates to a tangerine-derived bacterial cellulose gel for wound treatment and a dressing preparation containing the same, and more specifically, to reveal the wound healing effect of a cellulose gel extracted from citrus fruits using a gluconacetobacter strain, and a dressing containing the same. To provide sanctions.

The healing process of the wound can be divided into four stages: inflammatory, epithelial, proliferative and mature stages, but each stage is continuously overlapped to some extent without clear distinction. Of the four stages of wound healing, the most clinically important is the inflammatory stage. At this stage, destroyed tissue debris and necrotic tissue must be removed or incorporated by acute and chronic inflammatory cells to move on to the next stage, which lasts longer to delay the next wound healing process. Therefore, most of the materials or medicines used for wound healing help to hemostasis of wounds exposed to this inflammatory stage, block and protect the wound from harmful external environment, prevent microbial infection and heal quickly to the next wound healing stage. Its purpose is to make it progress. These materials also act in the proliferative phase after the inflammatory phase, which proliferates fibroblasts, activates the migration of fibroblasts, increases collagen synthesis, and ultimately promotes wound healing through wound contraction.

The dressing material must have high physical and mechanical durability to adhere to the wound to prevent wound protection and infection, have good biocompatibility, and the material itself can additionally exhibit pharmacological effects. In the meantime, materials that can be used as wound dressing materials have been extremely limited to materials that minimize rejection of biological tissues, that is, synthetic polymer classes having inertness to biological tissues. Natural materials have better human friendliness than synthetic materials, but rejection of the human body can not be completely excluded, and physical properties and mechanical strength are lower than those of synthetic polymer materials. In particular, it is pointed out that the natural polymer material is likely to be denatured according to the change of the surrounding environment, and the characteristics cannot be maintained uniformly.

Cellulose (cellulose) is a major component of the cell walls of higher plants and is a rich source of biological materials in nature. Plant cellulose is composed of heteropolysaccharides mixed with polysaccharides such as pectin, hemicelluloses, and lignin, resulting in low crystallinity and low mechanical strength and adsorption. Unlike the plant cellulose, the cellulose produced in the low polysaccharide content is relatively easy to purify and obtain pure cellulose. Therefore, cellulose extracted using bacteria has attracted attention as a new material in the food, cosmetics and pharmaceutical industries because of its excellent physical properties such as physical strength, water retention, crystallinity, adsorption, suspension stability and binding properties. In particular, bacterial cellulose has been shown to be effective in protecting and healing wet wounds due to its unique nanostructure, which is highly permeable and maintains proper humidity. In Germany and the United States, blood vessels and burns are present. It is a trend that is being developed as a medical product such as artificial skin (temporary skin) for patients.

As bacterial cellulose has attracted attention due to various industrial possibilities, much research has been done for mass production. In particular, when large quantities of bacterial cellulose are produced using surplus fruits, such as citrus fruits or coconuts, which are high in production, but with high yields, can be environmentally friendly and create high added value. Among them, citrus fruits are produced at 600,000 tons per year only in Jeju Island in Korea, and the consumption of processed products other than fresh fruit and citrus juice is low, and most of them are stored or discarded in concentrated liquid state. It is becoming.

In this regard, Republic of Korea Patent No. 1010055600000 'New Gluconacetobacter C1 strain and cellulose production method using the same' describes a method for producing cellulose using bacteria, but reports on how to use it industrially It has not been done.

1. Republic of Korea Patent Registration No. 1010055600000 (Registration date: December 27, 2010, Publication No. 1020090129068, Publication Date: December 16, 2009)

The present invention is to provide a cellulose gel and a dressing preparation containing the same using a gluconacetobacter strain, which has a remarkably superior wound healing effect compared to the prior art.

The present invention provides a wound treatment for cellulose gels produced using Gluconacetobacter sp. KCG-2 strain isolated from the natural fermentation broth of citrus juice.

In addition, the present invention provides a dressing agent for wound treatment containing the citrus-derived cellulose gel.

In the present specification, the term 'wound' does not merely mean a wound injured by a spear, bayonet, knife, or the like, but is used to encompass incisions, abrasions, surgical wounds, burns, ulcers, bedsores, infections or dermatitis after surgery. do.

The present invention provides a cellulose gel produced using Gluconacetobacter sp. KCG-326 strain isolated from the natural fermentation broth of citrus juice.

The cellulose gel according to the present invention may not only be used as a dressing material per se, but also may be applied to dressing materials such as bandages, adhesive bandages, bands, and mediforms.

In general, when the healing process of the wound is inhibited, the wound develops into a chronic wound, and the chronic wound is difficult to heal and often requires surgical treatment. Citrus-derived bacterial cellulose according to the present invention is effective in wound healing by reducing the initial inflammatory response and promoting the contractile reaction through the accumulation of extracellular matrix, and the dressing agent using the citrus-derived bacterial cellulose can be used as an effective wound healing agent.

1 is a photograph of the seventh day culture of citrus cellulose gel.
Figure 2 is a photograph showing a white cellulose gel after a purification process.
In Figure 3, A is a photo of the skin layer wound of 2 cm in diameter on three places including Sprague-Dawley species white paper, B is a vaseline gauze (a), alginate disk (b), a dressing material, Each of the cellulose gels according to the present invention is applied to photographs, and C is a photograph covered with 'Peha-haft® and sealed with nylon 4-0.
Figure 4 is a photograph of wound scars at 3, 6, 9, 12, 15, 18, 21, 24 and 28 days after the skin layer incision.
Figure 5 is a graph showing the wound area of the cellulose gel treatment group and petrolatum gauze treatment group according to the present invention, each bar represents the average value ± SE (n = 3) of three separate experiments, p <0.05 is petrolatum gauze It is considered to have a significant difference from the treatment group.
6 is a tissue result of staining the petrolatum gauze treatment group (left), Algisite M (registered trademark) treatment group (center) and the cellulose gel treatment group (right) according to the present invention 7 days after the procedure. Is a picture showing.
7 shows hematoxylin-eo in petrolatum gauze treatment group (top, left), Algisite M (registered trademark) treatment group (top, middle), cellulose gel treatment group (top, right) according to the present invention 14 days after the procedure. The result of the tissue stained with jeans is magnified 100 times, and below each is 200 times magnified.
Fig. 8 shows wound skin TGF-β and VEGF proteins of the petrolatum gauze treatment group (A), the alginate disc treatment group (B), and the cellulose gel treatment group (C) according to the present invention after 7 days, 14 days and 21 days after the wound. It is a photograph showing the expression, where actin was used as a control.

Although the present invention is described in more detail by the following examples, the following examples are for illustrative purposes only and it will be apparent to those skilled in the art that the scope of the present invention is not limited thereto.

Experimental material

Sprague-Dawley male white papers weighing 200g-250g were used as experimental animals. The experimental animals were kept under a breeding condition in which the contrast was automatically controlled at a temperature of 24 ± 0.5 ° C., a humidity of 55-65% and a 12 hour period, and used for experiments after adapting to the environment for about 14 days.

Manufacturing example  1: Citrus-derived Bacteria Cellulose Gel  Refining and fabrication

Gluconacetobacter sp. Gel_KCG326 strain, which is used to prepare cellulose gel, is a new strain isolated from citrus juice, and was deposited on May 11, 2010 to the Microbial Bank of Agricultural Genetic Resource Information Center. Number: KACC 91555P).

As a citrus concentrate used in the medium for gel fermentation, Seogwipo acid Wenju citrus concentrate with an average sugar content of 85 ° Brix was stored at -20 ° C and used after thawing.

As the gel fermentation medium, 9% of the commercial sugar solution, 1% (v / v) of the citrus concentrate, 1% (v / v) of ethanol, and 1% (v / v) of acetic acid were added to the citrus fruit juice medium for fermentation. After repeatedly culturing the gel_KCG326 strain of Gluconacetobacter sp. In the Gluconacetobacter sp., The gel formed on the surface of the medium was used as a seed. The medium was divided into 50 mL volumes of fermentation bottles by 35 mL, inoculated with 5% (W / V) of pre-incubated gel, and then the inlet of the fermentation bottles was covered with sterile filter paper and left incubated in 28 culture chambers for 7 days. As a result, the gel film was formed from the second day of culture, and the resulting citrus cellulose gel 7 days later is shown in FIG. 1.

Preparation Example 2 Purification of Citrus-derived Bacteria Cellulose Gel

Purification of the cellulose gel cultured in Preparation Example 1 was repeatedly treated with citrus gel 0.1N NaOH and 0.1N HCl to remove the pigment and impurities, and then dried in a 40 ℃ dry oven to remove moisture to the extent that it is suitable for dressing (dressing) After pressing, the mixture was pressurized and sterilized for 15 minutes. As a result, a white cellulose gel (thickness 2mm, diameter 26mm) suitable for dressing by removing the pigment, impurities and water was obtained (Fig. 2).

Experimental Example

(1) Preparation for Experiment

In order to investigate the wound healing effect of the citrus-derived bacterial cellulose gel according to the present invention, three round skin skin wounds having a diameter of 2 cm were made on white paper, and subdivided into A, B, C treatment groups according to the dressing material applied to each wound. The experimental group was prepared.

Vaseline gauze was used for the A-treated group. Vaseline gauze was selected as a representative material of the conventional dressing material, but it was used as a control because it keeps the wound wet but there is no growth factor or an ingredient that helps anti-inflammatory action.

In the treatment group B, Algisite M® nonwoven fabric (Smith & nephew, USA), which is known for its wound healing effect, was used.

In the C treatment group, the tangerine-derived bacterial cellulose gel prepared in the preparation example was used. Respectively.

(2) experimental process

Experiments were conducted with reference to reports that the delamination model was more suitable than the delamination model for measuring wound healing effects of topical treatments [Reimer, K., et al., Antimicrobial effectiveness of povidone-iodine and consequences for new application. areas . Dermatology, 2002. 204 Suppl 1: p. 114-20.].

Twenty white papers were made with three cutaneous cuticles of 2 cm diameter on each skin, and three dressing materials were randomly assigned to each wound (see FIG. 3). Experimental animals harvested skin tissue at the expense of four animals at 3, 7, 14, 21, and 28 days after surgery. At the time of surgery, anesthetized by intramuscular injection of zoletil 50 (Virbac, Frances), shaved the hair of white paper, disinfected with betadine and alcohol, and using the knife No. 15 and tissue scissors at the same size (2 cm in diameter). Epidermal wounds of the same depth (preservation of the panniculus carnosus, a superficial fascia that corrects the epidermal structure). In all treatment groups, dressings were done every other day until 7 days after surgery and once every 3-4 days from 8 days thereafter.

(3) experimental evaluation

1) Visual Evaluation

After the skin layer was made, the area of the wound was measured using a transparent OHP film sterilized and scanned and the area was measured using an image analysis program (NIH image analysis system, Bio-Optics, USA). . White skin was anesthetized and the area of the wound was measured every three days after the epidermal wound procedure. On the basis of the obtained values, the area was determined to be 100% on the first day of wound induction, and the change in area was compared.

As a result, as can be seen from Figure 4, a large amount of exudate was discharged from the wound site for 6 days after the wound, the exudate discharge rate in the treatment group dressing the cellulose gel according to the present invention decreases faster than the control group Vaseline gauze treatment group It was similar to the Algisite M® treatment group, which is known to be effective in wound healing. The wound area gradually decreased during the wound healing period, and most wounds healed between 18 and 28 days after surgery.

In addition, a graph showing the percentage of wound area decrease over time as a percentage with the initial wound area as 100% is shown in FIG. 5. As a result, the difference between the treated group in the petrolatum treated group (control group, 67.6%), Algisite M (registered trademark) treated group (60.4%) and cellulose gel treated group (64.3%) according to the present invention from 6 days after surgery Although not seen, the wound area was significantly reduced in all groups. Differences between treatment groups began to appear around 9 days after surgery. In the control group, the wound area was 50.2% on the 9th day after surgery, but in the cellulose gel treatment group according to the present invention, the difference was statistically 37.5%, and Algisite M (Trademark) treatment, which was proved to have a wound healing effect, was used. The figure was similar to that of the group (37.6%). On the 15th day after surgery, the wound area was 33.0%, 14.5% and 7.2% in the control group, Algisite M® group and citrus-derived bacterial cellulose gel group, respectively ( P <0.05). Appeared.

2) Histological observation

Wounds were made on white paper for histological evaluation, and then the entire wounds were removed on days 3, 7, 10, 14 and 21, immediately fixed in 10% formalin, embedded in paraffin, and cut into 6 μm thick hematoxylin-eozin. It was observed by staining with (Hematoxylin-Eosin). The effects of wound healing over time on each material were divided into four categories: re-epithelialization, fibroblast proliferation, multinucleated and lymphocyte infiltration, and angiogenesis.

On day 3 postoperatively, neutrophil infiltration was observed in all treatment groups and on day 7 postoperatively, neutrophil infiltration was increased in all treatment groups, but the petrolatum gauze treatment group was more effective than Algisite M® treatment group and cellulose gel treatment group according to the present invention. A large number of neutrophils were observed (see FIG. 6). In addition, the appearance of myofibroblasts was observed in all treatment groups, but the number of myofibroblasts in the Algisite M® treatment group and the cellulose gel treatment group according to the present invention was higher than that of the petrolatum gauze treatment group. Wound epithelialization was observed in all groups on day 14 postoperatively. Algisite M® and cellulose gel disc treatment groups according to the present invention had higher levels of collagen accumulation than petrolatum gauze treatment group, and the number of new blood vessels was observed. (See FIG. 7). However, the number of neovascularizations observed on day 14 postoperatively was less than on day 7 postoperatively.

3) Protein Analysis

Protein analysis was performed for TGF-β, which plays an important role in tissue regeneration and cell differentiation, and for VEGF, which is involved in angiogenesis. The tissue was placed in lysis buffer solution (iNtRON biotechnology, Korea), crushed with a tissue crusher, reacted on ice for 30 minutes, and centrifuged for 30 minutes (4 ° C., 12,000 rpm) to extract proteins. Protein concentration was quantified using the iNtRON biotechnology Protein assay kit, 50 mg of protein was denatured with 12% poly acrylamide gel electrophoresis (SDS-PAGE) and transferred to a nitrocellulose membrane (Amersham, Netherlands). , Blocking with TBS solution containing 5% skim milk at room temperature for 1 hour. Primary antibody transforming growth factor (TGF) -β, Vascular endothelial growth factor (VEGF) diluted in TBS (1: 1000) to measure protein expression (Santa Cruz, USA) was reacted at room temperature for 2 hours and then washed three times. Secondary antibodies are diluted 1: 2000 with anti-rabbit IgG, anti-mouse IgG (Santa Cruz, USA), reacted at room temperature for 1 hour, washed 5 times, and then reacted with ECL (Thermo, Japan) for photosensitization. The degree was confirmed.

The wound tissues extracted by the number of days in the wound model of the white paper were stored at -70 ℃, and the protein expression level of TGF-β1, an important growth factor involved in tissue regeneration and cell differentiation and VEGF, which influences angiogenesis after protein isolation Were analyzed by Western blotting (see FIG. 8). In the case of TGF-β1, the expression level was similar in all groups on the 7th day after surgery, but on the 14th and 21st day after the surgery, it was expressed only in the control group and treated with Algisite M® and the cellulose treatment group according to the present invention. Gradually decreased. VEGF was highest in citrus-derived bacterial cellulose treatment group and lowest in control group at 7 days after surgery, but after 14 days, it was expressed in TGF-β1 and high in control group and treated with Algisite M® treatment group. In the citrus-derived bacterial cellulose treatment group, it was gradually decreased. As shown in FIGS. 4 and 5, the growth factor expression was high at 7 days after the surgery when the wound healing was most active, and the expression level was lowered at 14 days when the wound was almost cured. On the other hand, the control group did not heal more than 40% of the wounds on the 12th day after surgery, and compared with the Algisite M® group and the citrus-derived bacterial cellulose group, the wound healing rate was slower. It was expressed higher than the group.

National Institute of Agricultural Science KACC91555 20100511

Claims (3)

Cellulose gel for wound treatment, characterized in that extracted from the tangerine using a gluconacetobacter strain. The dressing agent for wound treatment, wherein the cellulose gel of claim 1 is applied as an active ingredient. The dressing agent for wound treatment according to claim 2, which is in the form of a hydrocolloid or a wet foam.

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