Recombinant III-type collagen / platycladus orientalis essential oil nanoemulsion as well as preparation method and application thereof

Abstract

The invention relates to the technical field of biological agents, and provides a recombinant III-type collagen / platycladus orientalis essential oil nanoemulsion which is prepared from the following raw materials in percentage by weight: 15-35% of recombinant III-type collagen, 1-10% of platycladus orientalis essential oil and the balance of pure water. The invention also provides a preparation method of the nanoemulsion, which comprises the following steps: weighing the recombinant III collagen and the pure water according to the weight percentage, mixing and dissolving, then adding the platycladus orientalis essential oil, stirring and uniformly mixing to form primary emulsion, and carrying out high-speed shearing for 3-9 minutes by using a high-speed shearing emulsifying machine at the rotating speed of 8000r / min, so as to obtain the recombinant III collagen nanoemulsion. The recombinant III type collagen / platycladus orientalis essential oil nanoemulsion is obtained. The prepared oriental arborvitae essential oil nanoemulsion is uniform in size, high in stability and easy to store. The recombinant III type collagen has good biological activity and degradability, and can effectively reduce the irritation. The prepared nanoemulsion can significantly improve the antibacterial property of the platycladus orientalis essential oil nanoemulsion, and has a good prospect in application to bacterial infectious wounds.

Description

Technical Field

[0001] This invention relates to the technical field of biological agents, specifically to a method for preparing recombinant type III collagen / Thuja orientalis essential oil nanoemulsion and its application. Background Technology

[0002] Chinese arborvitae (Platycladus orientalis) is a tree belonging to the genus Platycladus of the family Cupressaceae. It is widely distributed and highly adaptable to various environments. Chinese arborvitae essential oil is an aromatic volatile compound extracted from arborvitae wood, containing mainly juniperol and thujone. Modern research has found that Chinese arborvitae essential oil has a certain inhibitory effect on Staphylococcus aureus and other bacteria. However, as a natural antibacterial agent, the poor water solubility, heat sensitivity, and volatility of Chinese arborvitae essential oil limit its application to some extent.

[0003] Type III collagen (COL3) is the second most abundant type of collagen in the body and plays a crucial role in fetal development and the maintenance and repair of tissues after birth. It can stimulate the proliferation and differentiation of fibroblasts, synthesize new collagen fibers, and fill wound defects.

[0004] Currently, commonly used wound care drugs in clinical practice have certain limitations, such as poor antibacterial effects, easy development of drug resistance, and high toxicity and irritation. Using recombinant type III collagen as an emulsifier and employing a high-speed shearing machine to prepare relatively stable arborvitae essential oil nanoemulsions can significantly improve the stability of arborvitae essential oil and reduce component changes caused by factors such as light and temperature. Furthermore, recombinant type III collagen has good biocompatibility and biodegradability, providing a favorable growth environment for cells. Arborvitae essential oil possesses antibacterial and anti-inflammatory biological activities, helping to reduce inflammation around wounds. Combining these two ingredients to prepare nanoemulsions can exert a synergistic effect; however, ensuring that the prepared nanoemulsions possess high stability and good antibacterial properties is one of the key technologies that needs to be addressed. Summary of the Invention

[0005] To address the aforementioned problems, this invention provides a recombinant type III collagen / Thuja orientalis essential oil nanoemulsion, its preparation method, and its applications. Recombinant type III collagen is non-toxic and low-irritant, and when combined with Thuja orientalis essential oil to replace traditional surfactants, the prepared recombinant type III collagen / Thuja orientalis essential oil nanoemulsion is uniform, stable, and enhances antibacterial effects, showing promising application prospects.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] The first aspect of the present invention provides a recombinant type III collagen / nanoemulsion, which is composed of the following raw materials in weight percentage: 15%-35% recombinant type III collagen, 1%-10% arborvitae essential oil, and the balance being pure water.

[0008] Furthermore, the nanoemulsion is obtained by high-speed shearing of recombinant type III collagen and arborvitae essential oil for 3-9 minutes. Recombinant type III collagen and arborvitae essential oil are the main active ingredients.

[0009] In some specific embodiments of the present invention, nanoemulsions prepared with different concentrations of Platycladus orientalis essential oil were investigated, wherein the concentration of Platycladus orientalis essential oil was 1%-10%, preferably 1%-5%, and more preferably 3%.

[0010] In some specific embodiments of the present invention, nanoemulsions prepared with different concentrations of recombinant type III collagen were examined, wherein the concentration of recombinant type III collagen was 15%-35%, preferably 20%-30%, and more preferably 25%.

[0011] Furthermore, the recombinant type III collagen / arborvitae essential oil nanoemulsion is composed of the following ingredients by weight percentage: 25% recombinant type III collagen, 3% arborvitae essential oil, and 72% pure water.

[0012] Furthermore, the nanoemulsion is obtained by high-speed shearing of recombinant type III collagen and arborvitae essential oil for 7 minutes.

[0013] A second aspect of this invention provides a method for preparing recombinant type III collagen / Thuja orientalis essential oil nanoemulsion, comprising the following steps:

[0014] (1) Dissolve recombinant type III collagen in pure water to obtain a collagen solution;

[0015] (2) Add arborvitae essential oil to the collagen solution and stir to mix well to form colostrum;

[0016] (3) The colostrum was subjected to high-speed shearing treatment using a high-speed shear emulsifier to obtain recombinant type III collagen / arborvitae essential oil nanoemulsion;

[0017] In the above preparation method, the recombinant type III collagen / arborvitae essential oil nanoemulsion contains, by weight percentage, 15%-35% recombinant type III collagen, 1%-10% arborvitae essential oil, and the remainder is pure water.

[0018] In the above preparation method, the high-speed shearing speed is 8000 r / min and the processing time is 3-9 min.

[0019] A third aspect of the present invention is an antibacterial drug whose active ingredient comprises the above-mentioned recombinant type III collagen / Thuja orientalis essential oil nanoemulsion.

[0020] A fourth aspect of this invention provides the application of the aforementioned recombinant type III collagen / Thuja orientalis essential oil nanoemulsion for the prevention of bacterial infection of wounds. The antibacterial activity verified in some specific embodiments of this invention demonstrates that, compared to aqueous solutions of Thuja orientalis essential oil, it exhibits anti-Staphylococcus aureus activity at lower concentrations. The recombinant type III collagen / Thuja orientalis essential oil nanoemulsion can be used in the preparation of antibacterial drugs.

[0021] The present invention has the following advantages over the prior art:

[0022] 1. This invention overcomes the shortcomings of arborvitae essential oil, such as poor water solubility and easy volatility. The prepared arborvitae essential oil nanoemulsion has uniform size and strong stability, and is easy to store. The prepared recombinant type III collagen / arborvitae essential oil nanoemulsion can significantly improve the dispersibility and antibacterial properties of arborvitae essential oil, and can effectively reduce the loss of arborvitae essential oil caused by volatility and photodecomposition.

[0023] 2. One of the raw materials of this invention, recombinant type III collagen, possesses excellent bioactivity and biodegradability. Using recombinant type III collagen as a surfactant effectively reduces irritation compared to traditional surfactants. Simultaneously, the prepared nanoemulsion significantly enhances the antibacterial properties of arborvitae essential oil nanoemulsion, making it a promising candidate for application in bacterial-infected wounds. Attached Figure Description

[0024] Figure 1 This is the GC-MS chromatogram of arborvitae essential oil;

[0025] Figure 2 The effect of different concentrations of Platycladus orientalis essential oil on the particle size and dispersibility of nanoemulsions;

[0026] Figure 3 The effect of different concentrations of recombinant type III collagen on nanoemulsion particle size and dispersion;

[0027] Figure 4 The effect of different shear times on nanoemulsion particle size and dispersion;

[0028] Figure 5 The effect of different concentrations of recombinant type III collagen / Thuja orientalis essential oil nanoemulsion solutions on the survival rate of Staphylococcus aureus;

[0029] Figure 6 The effect of each component of recombinant type III collagen / Thuja orientalis essential oil nanoemulsion on the survival rate of Staphylococcus aureus;

[0030] Figure 7 The growth curves of each component of the recombinant type III collagen / Thuja orientalis essential oil nanoemulsion against Staphylococcus aureus are shown.

[0031] Figure 8This is an experiment on the disruption of Staphylococcus aureus biofilm by various components of recombinant type III collagen / Thuja orientalis essential oil nanoemulsion;

[0032] Figure 9 The effects of each component of recombinant type III collagen / Thuja orientalis essential oil nanoemulsion on the morphology of Staphylococcus aureus;

[0033] In the accompanying diagram, the recombinant type III collagen group is abbreviated as collagen group, the arborvitae essential oil group is abbreviated as essential oil group, and the recombinant type III collagen / arborvitae essential oil nanoemulsion group is abbreviated as emulsion group. Detailed Implementation

[0034] The technical solution of the present invention will be further explained below with reference to embodiments. Those skilled in the art can implement the present invention using various other specific embodiments based on the content disclosed herein, or any simple changes or modifications made to the design structure and concept of the present invention will fall within the protection scope of the present invention. It should be noted that, in the absence of conflict, the embodiments and features in the embodiments of the present invention can be combined with each other.

[0035] In the following examples, the cypress essential oil was provided by Changzhou Yabo Fragrance Technology Co., Ltd., and the recombinant type III collagen was provided by Jiangsu Chuangjian Medical Technology Co., Ltd.

[0036] Example 1

[0037] 100 μL of Platycladus orientalis essential oil was dissolved in acetone and analyzed by gas chromatography-mass spectrometry (GC-MS). The analytical conditions were as follows: DB-17MS capillary column, 30 μm length, 0.25 mm inner diameter, 0.25 μm film thickness, and FID detector. The initial temperature was 100 °C (hold for 4 min), then increased to 180 °C at a rate of 10 °C / min, held for 5 min, and then increased to 230 °C at a rate of 5 °C / min, held for 2 min. The injection port temperature was 270 °C, the injection volume was 1.0 μL, splitless, helium carrier gas, and the flow rate was 1 mL / min. The mass spectrometer connection port temperature was 280 °C, the ion source temperature was 230 °C, the bombardment voltage was 70 eV, and the detection range was 15–500 μU. Figure 1 The image shows the GC-MS chromatogram of Platycladus orientalis essential oil. The GC-MS component analysis of Platycladus orientalis essential oil is shown in Table 1 below.

[0038] The component analysis in Table 1 shows that the content of juniper alcohol in arborvitae essential oil far exceeds that of other components. It is the main contributor to the woody and resinous aroma of the essential oil and also has bioactivity (such as calming, soothing, antibacterial, and antioxidant properties). It is a common raw material in the fields of daily chemical fragrances (such as perfumes and soaps) and natural medicines.

[0039] Table 1

[0040]

[0041] Example 2

[0042] Sample preparation: 0.25g of recombinant type III collagen was dissolved in 0.72g of purified water to obtain a collagen solution. 0.03g of Platycladus orientalis essential oil was weighed into a 2ml centrifuge tube. The collagen solution was added to the Platycladus orientalis essential oil and stirred until homogeneous to obtain a proemulsion. The proemulsion was subjected to high-speed shearing treatment at 8000r / min for 7 minutes using a high-speed shear emulsifier to obtain a recombinant type III collagen / Platycladus orientalis essential oil nanoemulsion. The thujone and thujone were present in relatively high proportions in the essential oil and are characteristic components of cypress family essential oils.

[0043] Example 3

[0044] The effects of different mass concentrations of Platycladus orientalis essential oil, recombinant type III collagen, and shear time on nanoemulsions were investigated.

[0045] Nanoemulsions were prepared using 1%, 3%, 5%, 7%, and 10% concentrations of Platycladus orientalis essential oil and 20% recombinant type III collagen, respectively, following the same preparation steps as in Example 1. The particle size distribution was analyzed using a Malvern particle size analyzer. Figure 2 As shown, nanoemulsions prepared with different concentrations of Platycladus orientalis essential oil have a significant impact on particle size and dispersibility. When the concentration of Platycladus orientalis essential oil is 3%, the nanoemulsion has the lowest particle size and dispersibility.

[0046] Nanoemulsions were prepared using recombinant type III collagen at concentrations of 15%, 20%, 25%, 30%, and 35%, and essential oil at a concentration of 3%, respectively. The preparation steps were the same as in Example 1, and the particle size distribution was analyzed using a Malvern particle size analyzer. Figure 3 As shown, with the increase of recombinant type III collagen concentration, the nanoemulsion particle size and dispersion exhibit a trend of first decreasing and then increasing. When the recombinant type III collagen concentration is 25%, the nanoemulsion particle size and dispersion are the lowest.

[0047] Nanoemulsions were prepared using recombinant type III collagen with shearing times of 3, 5, 7, and 9 minutes, along with 3% essential oil and 20% recombinant type III collagen. The preparation steps were the same as in Example 1. Analysis was performed using a Malvern particle size analyzer. Figure 4 As shown, with increasing shear time, the nanoemulsion particle size and dispersion exhibit a trend of first decreasing and then increasing. The nanoemulsion has the lowest particle size and dispersion when the shear time is 7 minutes.

[0048] Example 4

[0049] Investigating the antibacterial effect of recombinant type III collagen / Thuja orientalis essential oil nanoemulsion against Staphylococcus aureus.

[0050] Nanoemulsion solutions with concentrations of 10 mg / ml, 20 mg / ml, 30 mg / ml, 40 mg / ml, 50 mg / ml, and 60 mg / ml were prepared using purified water, with sterile water serving as the control group. S. aureus bacterial suspension in the logarithmic growth phase was diluted and mixed with aqueous solutions of concentrations of 5 mg / ml, 10 mg / ml, 15 mg / ml, 20 mg / ml, 25 mg / ml, and 30 mg / ml, and incubated on a shaker (250 rpm, 37℃) for 1 h. The volume of both the bacterial suspension and the nanoemulsion solution was 200 μL. The co-incubated sample was diluted 2 × 10⁻⁶ times. 4 Take 100 μL of the sample and spread it evenly on a solid agar plate. After 24 hours, count the bacteria and compare it with the blank control group to calculate the bacterial survival rate. Figure 5 As shown, the antibacterial effect was 95.1% when the concentration of the nanoemulsion solution was 15 mg / mL.

[0051] Example 5

[0052] The effects of various components of recombinant type III collagen / Thuja orientalis essential oil nanoemulsion on the survival rate of Staphylococcus aureus under the same conditions were studied. Nanoemulsion solutions with concentrations of 30 mg / mL, 0.9 mg / mL Thuja orientalis essential oil aqueous solution, and 7.5 mg / mL recombinant type III collagen solutions were prepared using ultrapure water, with sterile water serving as the control group. Logarithmic growth phase S. aureus bacterial suspension was diluted and incubated with nanoemulsion solutions at concentrations of 15 mg / mL, 0.45 mg / mL Thuja orientalis essential oil aqueous solution, and 3.75 mg / mL recombinant type III collagen solutions on a shaker (250 rpm, 37℃) for 1 h. The volume of both bacterial suspension and sample solutions was 200 μL. The incubated sample was diluted 2 × 10⁻⁶ times. 4 Take 100 μL of the sample and spread it evenly on a solid agar plate. After 24 hours, count the bacteria and compare it with the blank control group (i.e., the sterile water group) to calculate the bacterial survival rate. Figure 6 As shown, compared with the blank control group, the 0.45 mg / mL arborvitae essential oil solution had an inhibitory effect of about 62% on S. aureus, the 3.75 mg / mL recombinant type III collagen solution had no antibacterial effect on S. aureus, and the 15 mg / mL nanoemulsion aqueous solution had an inhibitory effect of more than 90% on S. aureus. Compared with the arborvitae essential oil aqueous solution, the antibacterial effect of arborvitae essential oil nanoemulsion was significant.

[0053] Example 6

[0054] The survival of various components of recombinant type III collagen / Thuja orientalis essential oil nanoemulsion against Staphylococcus aureus under comparable conditions was studied. Nanoemulsion solutions with concentrations of 30 mg / mL, Thuja orientalis essential oil aqueous solution with concentrations of 0.9 mg / mL, and collagen solutions with concentrations of 7.5 mg / mL were prepared using ultrapure water. Sterile water served as the control group. S. aureus bacterial suspension in the logarithmic growth phase was diluted 30-fold, and then the sample solutions were further diluted to concentrations of 15 mg / mL nanoemulsion solution, 0.45 mg / mL Thuja orientalis essential oil aqueous solution, and 3.75 mg / mL recombinant type III collagen solution. Samples were added to 96-well plates at 200 μL per well and incubated on a shaker (250 rpm, 37 °C). Absorbance was measured at 550 nm using a microplate reader every 1 hour. Figure 7 As shown, the study indicates that the collagen group had no antibacterial effect. Compared with the essential oil group, the recombinant type III collagen / Thuja orientalis essential oil nanoemulsion showed a significant antibacterial effect.

[0055] Example 7

[0056] The effects of different components of recombinant type III collagen / Thuja orientalis essential oil nanoemulsion on Staphylococcus aureus biofilm disruption under comparable conditions were investigated. S. aureus bacterial suspension in logarithmic growth phase was diluted 30-fold, and 200 μL of suspension was added to each well of a 96-well plate. After incubation in a bio-incubator for 48 h, the supernatant was aspirated using a 1 mL syringe. The sample wells were washed with PBS. Sample concentrations of 30 mg / mL nanoemulsion solution, 0.9 mg / mL Thuja orientalis essential oil aqueous solution, and 7.5 mg / mL recombinant type III collagen solution were diluted with TSB, with sterile water serving as a control. 150 μL of sample was added to each well of the 96-well plate and incubated for 2 h. The supernatant was aspirated, air-dried for 10 min, and then a suitable amount of crystal violet was added and allowed to stand for 10 min. After aspirating with a syringe, the plate was washed with PBS, and 100 μL of 80% ethanol was added. The plate was incubated on a shaker at constant temperature for 2 h, and the absorbance was measured at 590 nm using a microplate reader. Figure 8 As shown, compared with the essential oil group, the absorbance of the recombinant type III collagen / Thuja orientalis essential oil nanoemulsion was reduced, and it had a stronger destructive effect on the S. aureus biomembrane.

[0057] Example 8

[0058] The effects of different components of recombinant type III collagen / Thuja orientalis essential oil nanoemulsion on the morphology of Staphylococcus aureus under the same conditions were studied. One mL of logarithmic growth phase S. aureus bacterial suspension was placed in a 1.5 mL centrifuge tube and centrifuged at 4°C for 5000 rpm for 5 min. The bacterial suspension precipitate was resuspended in 500 μL of sample and incubated for 1 h. Then, it was centrifuged at 5000 rpm for 4°C for 5 min. The suspension was washed twice with PBS and centrifuged at 5000 rpm for 4°C for 5 min. The precipitate was then incubated in 200 μL of 4% glutaraldehyde in the dark for two hours and centrifuged at 5000 rpm for 4°C for 5 min. One mL of 5% ethanol was added, and the mixture was stirred until homogeneous. The solution was dehydrated for 10 min and centrifuged at 5000 rpm for 4°C for 5 min. The same procedure was repeated with 70% / 90% / 100% solutions for dehydration and centrifugation, followed by resuspending in anhydrous ethanol and adding the solutions dropwise to a silicon wafer to dry. Figure 9 As shown, the study indicates that the essential oil group and the recombinant type III collagen / Thuja orientalis essential oil nanoemulsion can alter the morphology of S. aureus. The bacterial membrane surface of the control group was smooth, while the bacterial membrane surface of the recombinant type III collagen / Thuja orientalis essential oil nanoemulsion group was more wrinkled.

[0059] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

Claims

1. A recombinant type III collagen / Thuja orientalis essential oil nanoemulsion, characterized in that, By weight percentage, it consists of the following components: 15%-35% recombinant type III collagen, 1%-10% arborvitae essential oil, and the balance being pure water.

2. The recombinant type III collagen / Thuja orientalis essential oil nanoemulsion according to claim 1, characterized in that, The content of the recombinant type III collagen is 20%-30%.

3. The recombinant type III collagen / Thuja orientalis essential oil nanoemulsion as described in claim 1, characterized in that, The content of the arborvitae essential oil is 1%-5%.

4. The recombinant type III collagen / Thuja orientalis essential oil nanoemulsion according to claim 1, characterized in that, By weight percentage, it consists of the following ingredients: 25% recombinant type III collagen, 3% arborvitae essential oil, and 72% pure water.

5. The method for preparing recombinant type III collagen / Thuja orientalis essential oil nanoemulsion according to any one of claims 1-4, characterized in that, The preparation method includes the following steps: (1) Dissolve recombinant type III collagen in pure water to obtain a collagen solution; (2) Add arborvitae essential oil to the collagen solution and stir to mix well to form colostrum; (3) The colostrum was subjected to high-speed shearing treatment using a high-speed shear emulsifier to obtain recombinant type III collagen / arborvitae essential oil nanoemulsion.

6. The preparation method according to claim 5, characterized in that, The recombinant type III collagen / arborvitae essential oil nanoemulsion contains, by weight percentage, 15%-35% recombinant type III collagen, 1%-10% arborvitae essential oil, and the remainder is pure water.

7. The preparation method according to claim 5, characterized in that, The high-speed shearing process has a rotation speed of 8000 r / min and a processing time of 3-9 minutes, preferably 7 minutes.

8. An antibacterial drug, characterized in that, Its active ingredients include the recombinant type III collagen / Thuja orientalis essential oil nanoemulsion as described in any one of claims 1-4.

9. The application of a recombinant type III collagen / Thuja orientalis essential oil nanoemulsion as described in any one of claims 1-4, characterized in that, Used to prevent bacterial infection of wounds.

10. The application according to claim 9, characterized in that, The nanoemulsion is used to enhance the antibacterial effect against Staphylococcus aureus and improve the quality of wound healing.

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