Application of a sesquiterpene alcohol compound as an ultraviolet sterilization synergist and method of the same

Abstract

This invention belongs to the technical field of ultraviolet (UV) sterilization, specifically disclosing the application of sesquiterpene alcohols as UV sterilization synergists and their combined UV sterilization method. This invention discovers that sesquiterpene alcohols, such as valerian oil, have a synergistic antibacterial effect with UV light. In vitro combined sterilization studies have shown that the bacterial count is reduced by at least 2-3 Log values ​​compared to individual administration, and the sterilization effect is significantly enhanced compared to the individual action of UVA and valerian oil. Compared to traditional UV sterilization technologies, this invention can kill bacteria more efficiently and reduce the impact of UV lamp quality on disinfection effectiveness.

Description

Technical Field

[0001] This invention relates to the technical field of ultraviolet sterilization, and specifically discloses the application of sesquiterpene alcohols as ultraviolet sterilization synergists and the method of combining them with ultraviolet sterilization. Background Technology

[0002] Ultraviolet (UV) sterilization is a simple, chemical-residue-free sterilization method that is unlikely to induce drug resistance. It is commonly used to disinfect gases, liquids, and solid surfaces. Ultraviolet radiation is a general term for radiation with wavelengths between 10 nm and 400 nm in the electromagnetic spectrum. According to wavelength, UV radiation is classified into UVA, UVB, UVC, and UVD. In recent years, the widespread use of antibiotics has led to increasingly serious bacterial resistance, accompanied by the emergence of multidrug-resistant bacteria. This has resulted in a gradual weakening of the effectiveness of antibiotics in treating infections. Therefore, research into non-antibiotic sterilization techniques is increasingly being conducted in clinical practice, and research on the effectiveness of UV sterilization is becoming more in-depth.

[0003] The principle of ultraviolet (UV) sterilization technology is to utilize ultraviolet radiation of appropriate wavelengths to destroy the molecular structure of DNA (deoxyribonucleic acid) or RNA (ribonucleic acid) within biological cells, causing the death of growing and / or regenerating cells, thus achieving a sterilization effect. UV sterilization technology is based on modern epidemic prevention science, medicine, and photodynamics. It uses specially designed, highly efficient, high-intensity, and long-life UV light to irradiate the surface of objects, directly killing various bacteria, viruses, parasites, algae, and other pathogens on the surface. It can kill bacteria without inducing antibiotic resistance, thus preventing the increase of bacterial resistance. However, low-energy UV light itself is insufficient to achieve a very good sterilization effect, the sterilization time is long, and it cannot completely kill bacteria, especially with limited effectiveness against some drug-resistant strains, restricting its application in high-end disinfection scenarios.

[0004] To enhance the bactericidal effect of ultraviolet (UV) radiation, reduce UV irradiation dose, and shorten sterilization time, researching UV sterilization synergists is an essential step. However, currently reported synergistic effects of essential oils on UV sterilization are still limited, necessitating the development of new UV sterilization synergists to increase the bactericidal effect of UV radiation in various situations. Sesquiterpenoids possess various biological activities, including anti-inflammatory, antibacterial, and antioxidant properties, with some exhibiting anticancer and anti-inflammatory potential. Valerian oil, extracted from the roots, stems, and leaves of Valerianaceae (Valeraria genus) through distillation, belongs to the sesquiterpenoid class of compounds. Due to its unique sedative and tranquilizing effects and natural antibacterial activity, it has been widely used in medicine, daily chemicals, and food preservation in recent years. However, the application of valerian oil in combination with UV sterilization has not yet been reported. Summary of the Invention

[0005] To address the shortcomings of existing technologies, the primary objective of this invention is to provide an application of sesquiterpene alcohol compounds as ultraviolet bactericidal synergists.

[0006] The second objective of this invention is to provide a method for sterilization using sesquiterpene alcohols in conjunction with ultraviolet light.

[0007] To achieve the above objectives, the present invention provides the following technical solution:

[0008] Application of sesquiterpene alcohols as UV bactericidal synergists.

[0009] Preferably, the sesquiterpene alcohol compound is valerian oil.

[0010] Valerian oil (CAS: 8008-88-6, the valerian oil used in this invention was purchased from Maclean's, catalog number V854433) has the following chemical structural formula: .

[0011] Valerian oil belongs to the sesquiterpene alcohol class of compounds. Due to its natural, non-toxic, biodegradable, and residue-free properties, it holds great promise as a natural antibacterial agent and disinfectant synergist. This invention utilizes the natural antibacterial properties of valerian oil to make it a superior candidate material for ultraviolet (UV) bactericidal synergist. Experimental studies have shown that the combined use of valerian oil and UV light significantly enhances UV bactericidal activity, thus making it a suitable synergist for UV sterilization.

[0012] The present invention also provides an ultraviolet bactericidal synergist, wherein the synergist comprises sesquiterpene alcohols.

[0013] Preferably, the sesquiterpene alcohol compound is valerian oil.

[0014] The present invention also provides a method for sterilization by combining sesquiterpene alcohols with ultraviolet light.

[0015] Preferably, the sesquiterpene alcohol compound is valerian oil with a concentration of 1.25%; and the ultraviolet irradiation time is 30 min.

[0016] Preferably, the ultraviolet intensity in the above sterilization method is 2.4~2.5 mW / cm². 2 .

[0017] Preferably, in the above sterilization method, preheating for 30 to 60 minutes is required before ultraviolet irradiation.

[0018] Preferably, the above-mentioned sterilization method includes the following steps:

[0019] S1. Place the bacterial culture in a six-well plate and mix it thoroughly with valerian oil. Set the UV irradiation conditions as follows: wavelength range 300~469 nm, power 16W, distance from the six-well plate to the UV lamp tube 8cm, and UV intensity 2.4~2.5 mW / cm². 2 ;

[0020] S2. Preheat the UV chamber for 30-60 minutes, then place the six-well plate in the UV chamber and irradiate for 30 minutes.

[0021] More preferably, the bacterial solution in step S1 is Escherichia coli BW25113.

[0022] More preferably, the final concentration of the bacterial solution in step S1 is 10. 6 CFU / mL.

[0023] Compared with the prior art, the beneficial effects of the present invention are:

[0024] This invention provides valerian oil, a sesquiterpene alcohol compound with synergistic bactericidal effects with ultraviolet (UV) light. The invention reveals that valerian oil and UVA exhibit a synergistic bactericidal effect. In vitro combined bactericidal experiments demonstrate that the combined use of the two reduces bacterial count by at least 2-3 Log values ​​compared to either agent alone, and significantly enhances the bactericidal effect compared to the individual effects of valerian oil and UVA. Compared to traditional UV sterilization technologies, this invention can kill bacteria more efficiently and reduce the impact of UV lamp quality on disinfection effectiveness. Attached Figure Description

[0025] Figure 1 This is a graph showing the statistical results of the number of strains in Example 4. Detailed Implementation

[0026] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0027] Unless otherwise specified, the experimental methods used in the embodiments of this invention are conventional methods; unless otherwise specified, the materials and reagents used are commercially available.

[0028] The sources of commercially available medicinal extracts are as follows, and do not constitute a limitation of this invention: The ultraviolet lamp used was a PHILIPS TL-D brand; wattage: 16W; voltage: 220V; wavelength: (UVA) 300-469nm; lamp tube diameter: 25mm; length: 60cm. The valerian oil used was a Maclean brand (China, Shanghai, product number V854433). The Escherichia coli BW25113 used was purchased from Beijing Bio-Bio Biotechnology Co., Ltd.

[0029] Synergistic judgment principle: If the bacterial count reduction in the experimental group is two log reductions on top of the bacterial count reductions in UV treatment alone and valerian oil treatment alone, it indicates that valerian oil and UV have a synergistic bactericidal effect.

[0030] Example 1: Bactericidal effect of different UV irradiation times on strain BW25113

[0031] 1. Experimental materials:

[0032] (1) The UV lamp used in the experiment was a PHILIPS TL-D brand; wattage: 16W; voltage: 220V; wavelength: (UVA) 365nm; tube diameter: 25mm; length: 60cm.

[0033] (2) Experimental culture medium: LB agar medium and MacConkey medium (purchased from Guangdong Huankai Microbial Technology Co., Ltd.) that have been autoclaved are cooled to 45°C, and 20 mL is taken with a pipette and placed into a sterile culture dish. The mixture is then air-dried for 30 min to obtain LB agar medium and MacConkey medium.

[0034] Escherichia coli standard strain BW25113 (laboratory collection).

[0035] 2. Preparations before the experiment:

[0036] (1) Turn on the ultraviolet lamp and irradiate continuously for 30 minutes to preheat, keeping the temperature at 36℃~38℃;

[0037] (2) The standard strain of Escherichia coli BW25113 was cultured on streak resurrection MacConkey medium until it reached the appropriate size.

[0038] 3. Evaluation experiment on the bactericidal effect of ultraviolet light:

[0039] (1) Inoculate a single colony of Escherichia coli BW25113 into a centrifuge tube containing 4 mL of LB broth, place it in a 37°C shaker and incubate at 180 rpm for 4 hours, then remove the centrifuge tube.

[0040] (2) Place the centrifuge tubes in a centrifuge, centrifuge at 5500 rpm for 5 minutes, discard the supernatant, add an equal volume of physiological saline to resuspend, and perform serial dilution to achieve a final bacterial count of 10. 6 CFU / mL;

[0041] (3) Add 1 mL of bacterial culture to a six-well plate;

[0042] (4) Set up a blank control group and an ultraviolet irradiation treatment group. The ultraviolet irradiation treatment group was divided into three groups, which were irradiated for 30 min, 60 min and 90 min respectively.

[0043] (5) After UV irradiation, 100 μL of bacterial culture was added to a 2 mL sterile centrifuge tube containing 900 μL of physiological saline for serial dilution. 25 μL of the diluted culture was then dropped onto LB agar medium and incubated at 37°C for 16-18 h. Bacterial counts were then performed, and the results were statistically analyzed after three biological replicates. The results are shown in Table 1. With increasing irradiation time, the antibacterial effect of UV on bacteria increased. However, in the 30 and 60 min UV irradiation groups, the bacterial count was not significantly different from the control group, indicating that short UV irradiation times do not produce a significant antibacterial effect on BW25113. In the 90 min UV irradiation group, the bacterial count began to decrease, indicating that UV began to have a certain antibacterial effect.

[0044] Table 1. Effect of UV irradiation time on the bactericidal activity of strain BW25113

[0045] Example 2: Determination of the MIC of different concentrations of valerian oil against strain BW25113

[0046] 1. Experimental materials:

[0047] (1) Experiment: The MH broth, which has been autoclaved, was cooled and set aside for later use.

[0048] (2) Resin Azure: Macklin brand, MW 251.17, purity 90%.

[0049] Escherichia coli standard strain BW25113 (laboratory collection).

[0050] 2. Preparations before the experiment:

[0051] (1) Remove the valerian oil from the refrigerator and allow it to reach room temperature. Take a portion of the oil and set it aside.

[0052] (2) Streaking Escherichia coli standard strain BW25113 onto MacConkey agar medium and culturing until it reaches the appropriate size;

[0053] (3) Weigh 0.2512g of resin azurite powder into a centrifuge tube, add 10mL of pure water to dissolve it, and the concentration of the resin azurite solution is 10mM / L.

[0054] 3. Evaluation experiment on the bactericidal effect of valerian oil:

[0055] (1) Inoculate a single colony of Escherichia coli BW25113 into a centrifuge tube containing 4 mL of MH broth, incubate at 180 rpm for 4 hours in a 37°C shaker, and then remove the centrifuge tube;

[0056] (2) Dilute the incubated E. coli 100 times with MH broth, the bacterial count is approximately 10. 6 CFU / mL, for later use;

[0057] (3) Take a sterile 96-well plate, add 180 μL of MH broth medium to well 1, and add 100 μL of MH broth medium to wells 2-11;

[0058] (4) Add 20 μL of the original concentration of essential oil to the first well, blow it evenly, and then draw 100 μL into the second well, and so on, drawing 100 μL from the tenth well and discarding it; at this time, the concentration of essential oil in each well is 10%, 5%, 2.5%, 1.25%, 0.625%, 0.3125%, 0.156%, 0.078%, 0.039%, and 0.0195%;

[0059] (5) Add 100 μL of diluted bacterial solution to wells 1 to 10. At this time, the essential oil concentrations in each well are 5%, 2.5%, 1.25%, 0.625%, 0.3125%, 0.156%, 0.078%, 0.039%, 0.0195%, and 0.01%, respectively. Add 100 μL of bacterial solution to well 11 (L) and 200 μL of MH broth to well 12.

[0060] (6) Repeat steps (3) to (5) to set up three parallel groups;

[0061] (7) After incubating the inoculated 96-well plate in a 37°C incubator for 16-18 hours, add 10 μL of 10 mM / L azuril resin to each well (resin concentration: 0.1 mM / L). Incubate for 2 hours and then read the results. The results are shown in Table 2. The MIC value of valerian oil was 1.25%, at which bacterial growth was inhibited. Therefore, a concentration of 1.25% valerian oil was ultimately selected as the concentration for use as the UV synergist.

[0062] Table 2. MIC and experimental concentration of valerian oil against strain BW25113

[0063] Example 3: The bactericidal effect of valerian oil on strain BW25113 after 30 minutes of contact.

[0064] 1. Experimental materials:

[0065] (1) Experimental culture medium: LB agar medium and MacConkey agar medium (purchased from Guangdong Huankai Microbial Technology Co., Ltd.) that have been autoclaved are cooled to 45°C, and 20 mL is taken with a pipette and put into a sterile culture dish. The mixture is then air-dried for 30 min to obtain LB agar medium and MacConkey agar medium.

[0066] Escherichia coli standard strain BW25113 (laboratory collection).

[0067] 2. Preparations before the experiment:

[0068] (1) Remove the valerian oil from the refrigerator and allow it to reach room temperature. Take a portion of the oil and set it aside.

[0069] (2) The standard strain of Escherichia coli BW25113 was streaked onto MacConkey medium and cultured until it reached the appropriate size.

[0070] 3. Evaluation experiment on the bactericidal effect of valerian oil:

[0071] (1) Inoculate a single colony of Escherichia coli BW25113 into a centrifuge tube containing 4 mL of LB broth, place it in a 37°C shaker and incubate at 180 rpm for 4 hours, then remove the centrifuge tube.

[0072] (2) Place the centrifuge tubes in a centrifuge, centrifuge at 5500 rpm for 5 minutes, discard the supernatant, add an equal volume of physiological saline to resuspend, and perform serial dilution to achieve a final bacterial count of 10. 6 CFU / mL;

[0073] (3) Add 1 mL of bacterial culture to a six-well plate, add 12.5 μL of the original concentration of valerian oil and mix well to make the final concentration (V / V) of essential oil 1.25%;

[0074] (4) Set up controls: blank control and 1.25% valerian oil group treated for 30 min;

[0075] (5) Take 100L of bacterial culture and add it to a 2ml sterile centrifuge tube containing 900μL of physiological saline for serial dilution. After dilution, take 25μL and drop it onto LB agar medium. Incubate at 37 degrees Celsius for 16-18h and count the results. The experimental results are statistically analyzed after three biological replicates.

[0076] The results are shown in Table 3. There was no statistically significant difference in bacterial count between the blank control group and the group treated with valerian oil for 30 minutes; indicating that 1.25% valerian oil did not cause any change in bacterial count after 30 minutes of treatment with BW25113 bacteria. Therefore, 1.25% valerian oil alone did not have a significant antibacterial effect on BW25113 bacteria.

[0077] Table 3. Bactericidal effect of valerian oil on strain BW25113

[0078] Example 4 Evaluation of the bactericidal effect of ultraviolet light and ultraviolet synergists on Escherichia coli BW25113

[0079] 1. Experimental materials:

[0080] (1) The UV lamp used in the experiment was a PHILIPS TL-D brand; wattage: 16W; voltage: 220V; wavelength: (UVA) 300-469nm; tube diameter: 25mm; length: 60cm.

[0081] (2) Experimental culture medium: LB agar medium and MacConkey agar medium (purchased from Guangdong Huankai Microbial Technology Co., Ltd.) that have been autoclaved are cooled to 45°C. 20 mL of each medium is taken with a pipette and placed into a sterile culture dish. The medium is then air-dried for 30 min to obtain LB agar medium and MacConkey agar medium.

[0082] Escherichia coli standard strain BW25113 (laboratory collection).

[0083] 2. Preparations before the experiment:

[0084] (1) Turn on the ultraviolet lamp and irradiate continuously for 30 minutes to preheat:

[0085] (2) Remove the valerian oil from the refrigerator and allow it to reach room temperature. Extract a portion for later use:

[0086] (3) Incubate the standard strain of Escherichia coli BW25113 on MacConkey agar medium until it reaches the appropriate size.

[0087] 3. Evaluation experiment on the effect of ultraviolet synergist:

[0088] (1) Inoculate a single colony of Escherichia coli BW25113 into a centrifuge tube containing 4 mL of LB broth, incubate at 37 degrees Celsius and 180 rpm for 4 hours, then remove the centrifuge tube.

[0089] (2) Place the centrifuge tubes in a centrifuge, centrifuge at 5500 rpm for 5 minutes, discard the supernatant, add an equal volume of physiological saline to resuspend, and perform serial dilution to achieve a final bacterial count of 10. 6 CFU / mL;

[0090] (3) Add 1 mL of bacterial culture to a six-well plate, add 12.5 μL of the original concentration of valerian oil and mix well;

[0091] (4) Set up a blank control group, an essential oil treatment group, a UVA group, and a combination group;

[0092] (5) The UV device configured as described above uses 2.4~2.5mW / cm².2 After 30 min of UV intensity treatment, 100 μL of bacterial culture was added to a 2 ml sterile centrifuge tube containing 900 μL of physiological saline for serial dilution. After dilution, 25 μL was dropped onto LB agar medium and incubated at 37 degrees Celsius for 16-18 h. The bacterial culture was counted, and the experimental results were statistically analyzed after three biological replicates.

[0093] In this experiment, a growth control group, a UV control group, an essential oil control group, and a UV plus essential oil test group were set up, and the tests were carried out according to the method in Example 1.

[0094] Table 4. Bactericidal effect of combined ultraviolet light and valerian oil on strain BW25113

[0095] The results are shown in Table 4. The bacterial count in the blank control group showed only slight changes, indicating that Escherichia coli BW25113 could survive normally under these experimental conditions. After 30 minutes of UV irradiation, the number of Escherichia coli BW25113 was not significantly reduced compared to the blank control group, indicating that UV radiation had little inhibitory effect on Escherichia coli BW25113. Similarly, the results also showed that 1.25% valerian oil had little inhibitory effect on Escherichia coli BW25113. The combined effect of UV radiation and 1.25% valerian oil for 30 minutes showed that the combined effect of UV radiation and 1.25% valerian oil on Escherichia coli BW25113 was significant and had a synergistic bactericidal effect.

[0096] Obviously, the specific implementation schemes described above are merely a further detailed explanation of the purpose, technical solution and beneficial effects of the present invention. It should be understood that the above descriptions are only specific examples of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. The application of sesquiterpene alcohols as ultraviolet bactericidal synergists, characterized in that, The sesquiterpene alcohol compound is valerian oil.

2. A UV bactericidal synergist, characterized in that, The synergist includes sesquiterpene alcohols such as valerian oil.

3. A method for sterilization using sesquiterpene alcohols in combination with ultraviolet light, characterized in that, The sesquiterpene alcohol compound was valerian oil with a concentration of 1.25%, and the ultraviolet irradiation time was 30 min.

4. The method according to claim 3, characterized in that, The ultraviolet intensity is 2.4~2.5 mW / cm. 2 .

5. The method according to claim 4, characterized in that, Preheating for 30 to 60 minutes is required before ultraviolet irradiation.

6. The method according to claim 5, characterized in that, Includes the following steps: S1. Place the bacterial culture in a six-well plate and mix it thoroughly with valerian oil. Set the UV irradiation conditions as follows: wavelength range 300-469nm, power 16W, distance from the six-well plate to the UV lamp tube 8cm, and UV intensity 2.4~2.5mW / cm². 2 ; S2. Preheat the UV chamber for 30-60 minutes, then place the six-well plate in the UV chamber and irradiate for 30 minutes.

7. The method according to claim 6, characterized in that, The bacterial solution mentioned in step S1 is Escherichia coli BW25113.

8. The method according to claim 7, characterized in that, The final concentration of the bacterial solution in step S1 is 10. 6 CFU / mL.

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