Disinfectant for use in photodynamic therapy

Abstract

To provide a novel bactericide for use in photodynamic therapy.SOLUTION: According to the present invention, there is provided a bactericide for use in photodynamic therapy, which contains xanthohumol as an active ingredient. When the bactericide of the present invention is used at a site to which photodynamic therapy is to be applied, an extremely strong bactericidal effect can be obtained. The bactericide of the present invention can be used, for example, for dental treatment or dermatological treatment. The present invention also provides a sterilization method by photodynamic therapy, which is characterized by carrying out light irradiation in the presence of xanthohumol.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] This invention relates to a bactericide for use in photodynamic therapy. [Background technology]

[0002] In dental treatment, particularly in root canal treatment (endodontic treatment), the damaged pulp is removed (pulpectomy), the root canal is carefully cleaned, and measures are taken to prevent reinfection. Sterilization of the root canal is essential in root canal treatment for pulpitis, where caries progresses and infection reaches the pulp, and apical periodontitis, where the infection spreads outside the apical foramen. However, the inside of the root canal has a very fine and complex structure, and it is known that bacteria can easily remain even after mechanical and chemical cleaning (Non-patent Literature 1).

[0003] Sodium hypochlorite solution is often used for root canal irrigation, but complete sterilization is often difficult. In contrast, the application of the instantaneous thermal effect of a laser is being considered as an effective treatment method for sterilizing the inside of the root canal. By using a laser, bacteria remaining in the root canal can be efficiently killed, thereby enhancing the treatment effect. However, while the sterilization ability increases with higher laser power, the damage to surrounding tissues also increases. For this reason, photodynamic therapy (PDT), which uses a minimally invasive light source and a photosensitive substance with low tissue invasiveness, has been proposed. The antibacterial effect against caries bacteria using a diode laser with a wavelength of 660 nm and methylene blue as a photosensitive substance has been reported (Non-Patent Literature 2), and in Japan, the use of PDT is being introduced in dentistry as a private treatment not covered by insurance, using combinations of methylene blue and a diode laser (650-675 nm), or toluidine blue and an LED (620-640 nm).

[0004] However, while methylene blue has a relatively high safety profile, there are concerns about its side effects on the blood system, and there is currently little clear information regarding the safety of toluidine blue, so concerns remain about its clinical use. Against this backdrop, a bactericide for photodynamic therapy using an alkaline extract of Sasa veitchii as a photosensitive substance (Patent Document 1) and a bactericidal method involving light irradiation of catechins (Patent Document 2) have been reported. [Prior art documents] [Patent Documents]

[0005] [Patent Document 1] Japanese Patent Publication No. 2021-116253 [Patent Document 2] International Publication No. 2012 / 098772 [Non-patent literature]

[0006] [Non-Patent Document 1] Vera J. et al, J. Endod, 38, 1044-1052, 2012 [Non-Patent Document 2] Stojic S et al., Int. Endod. J., 46, 649-659, 2013 [Overview of the project] [Problems that the invention aims to solve]

[0007] The present invention aims to provide a novel bactericide for use in photodynamic therapy. [Means for solving the problem]

[0008] The inventors of this invention have been diligently conducting research focusing on ingredients and materials that are contained in food and beverages, have a long history of oral ingestion, and have a high level of safety. They discovered that applying xanthohumol to photodynamic therapy enhances the bactericidal effect of photodynamic therapy, achieving an extremely strong bactericidal effect. This invention is based on this finding.

[0009] The present invention provides the following inventions. [1] A disinfectant containing xanthohumol as the active ingredient, used in photodynamic therapy. [2] The disinfectant described in [1] above for enhancing the bactericidal effect of photodynamic therapy. [3] The disinfectant described in [1] or [2] above, for use in dental treatment. [4] The disinfectant described in [3] above, for dental treatment being root canal treatment. [5] The disinfectant described in [1] or [2] above, for use in dermatological treatment. [6] The fungicide described in [5] above, wherein the dermatological treatment involves inhibiting or killing the growth of the causative bacteria. [7] A disinfectant according to any of [1] to [6] above, which is applied to the light-irradiated area so that the concentration of xanthohumol is 0.1 mM to 100 mM. [8] A photodynamic sterilization method characterized by light irradiation in the presence of xanthohumol. [9] The method described in [8] above, which is an adjunct to dental or dermatological treatment.

[0010] According to the present invention, it is possible to provide a disinfectant for photodynamic therapy that can exert an extremely strong bactericidal effect and can be used safely. [Brief explanation of the drawing]

[0011] [Figure 1] Figure 1 shows the results (Experiment 1 and Experiment 2) of evaluating the bactericidal effect of E. faecalis bacterial suspension when irradiated with a laser in the presence of (A) DMSO (solvent: control), (B) resveratrol, and (C) xanthohumol, respectively, and when not irradiated with a laser. Detailed description of the invention

[0012] The bactericide of the present invention is a bactericide for use in photodynamic therapy. Here, photodynamic therapy refers to a therapy in which a photosensitive substance is applied to a target site of a living body, and light of a predetermined wavelength is irradiated onto the target living tissue, thereby exciting the photosensitive substance to generate molecular species having bactericidal properties such as radicals and reactive oxygen species, and achieving a bactericidal effect.

[0013] The bactericide of the present invention contains xanthohumol as an active ingredient.

[0014] Xanthohumol, which is the active ingredient of the present invention, is a kind of flavonoid contained in hops ( Humulus lupulus ), and is a compound registered with the CAS number: 6754-58-1. Xanthohumol can be prepared from hop cones by a plurality of known methods, for example. According to JP-A-2007-289185, it is said that a powder containing xanthohumol with high purity can be produced. There is also a report that xanthohumol can be efficiently purified by CCC (counter-current chromatography) (Qi-He Chen, Ming-Liang Fu, Miao-Miao Chen, Jing Liu, Xiao-Jie Liu, Guo-Qing He, Shou-Cheng Pu, Food Chem, 132(1):619-23 (2012)). That is, in the present invention, xanthohumol derived from hops can be used as an active ingredient, and in this case, xanthohumol may be in any form of isolated, purified or roughly purified. In the present invention, a synthetic product can also be used as an active ingredient as xanthohumol.

[0015] Xanthohumol, which is the active ingredient of the present invention, can also be used in the form of a salt. For example, it can be used in the form of a salt coordinated with metal ions such as pharmaceutically acceptable aluminum, iron, copper, calcium, etc. (for example, WO 2020 / 212882).

[0016] The bactericide of the present invention may contain any component other than xanthohumol. Examples of the optional component include pharmaceutically acceptable formulation additives for preparing xanthohumol as a formulation and reaching the target site. The bactericide of the present invention can be produced by mixing xanthohumol and an optional component.

[0017] In the present invention, xanthohumol and other bactericidal components may be used in combination. Examples of such bactericidal components include photosensitive substances used in photodynamic therapy (see, for example, Patent Document 1). Further, the bactericide of the present invention may be used in combination with other bactericidal components other than xanthohumol.

[0018] As shown in the following examples, when the bactericide of the present invention is used in photodynamic therapy, an extremely strong bactericidal effect can be obtained. Therefore, the bactericide of the present invention can be used to enhance the bactericidal effect by photodynamic therapy.

[0019] Since the bactericide of the present invention exhibits an extremely strong bactericidal action in photodynamic therapy, it can be used for oral disinfection and can be used for dental treatment aimed at treating periodontal disease, gingivitis, dental caries, etc. The bactericide of the present invention can also be used for the disinfection of tissues and organs other than the oral cavity. The bactericide of the present invention can be used, for example, for dermatological treatment (for example, treatment of acne vulgaris (pimples), etc.). Non-limiting examples of dermatological treatment include the inhibition of growth or sterilization of causative bacteria (for example, acne bacteria) of skin diseases such as acne vulgaris.

[0020] When the bactericide of the present invention is used for dental treatment, it can be used as follows. That is, as described above, in dental treatment, especially in root canal treatment, sterilization of the root canal is essential. However, the root canal has a very fine and complex structure, and it is known that bacteria tend to remain even after mechanical and chemical cleaning. Although bactericidal cleaning of the root canal with such a complex form is performed in dental treatment using photodynamic therapy, the bactericide of the present invention can extremely strongly enhance the bactericidal effect by photodynamic therapy, and thus can be suitably used for dental treatment.

[0021] When using the bactericide of the present invention in photodynamic therapy, the light source can be a laser, halogen lamp, LED, etc. The laser is not particularly limited as long as it can produce light of the wavelength necessary for excitation, and examples include diode lasers, dye lasers, argon lasers, etc., as long as it is a laser used for medical purposes. When using a laser as the light source, Nd:YAG lasers, Er:YAG lasers, carbon dioxide lasers, etc. can be used. If an LED is used, any medically used LED such as a red LED (approximately 630 nm), blue LED (approximately 470 nm), or yellow-green LED (approximately 565 nm) is acceptable.

[0022] The wavelength of the light ray can be determined by considering the absorption wavelength in photodynamic therapy, taking into account the maximum absorption wavelength of xanthohumol (368 nm), the maximum absorption wavelengths of other bactericidal components, and the wavelength of the medical laser. In this invention, the wavelength of the light ray can be, for example, in the range of 300 nm to 700 nm (preferably 300 nm to 600 nm, more preferably 350 nm to 500 nm).

[0023] The irradiation time and light output can be appropriately adjusted by those skilled in the art to obtain the desired sterilization effect. In the present invention, for example, the light can be irradiated for 30 seconds or more, preferably 60 seconds or more, and the light output can be 3W or more. The upper limit of the irradiation time can be, for example, 180 seconds or 120 seconds. The upper limit of the light output can be, for example, 10W.

[0024] The bactericidal agent of the present invention can be applied to the light-irradiated site at a xanthohumol concentration of, for example, 0.1 mM to 100 mM (preferably 1 mM to 100 mM, more preferably 1 mM to 50 mM). If the bactericidal agent of the present invention is a composition with a high xanthohumol content, it can be used after being appropriately diluted.

[0025] According to another aspect of the present invention, a method for sterilization by photodynamic therapy is provided, characterized by performing light irradiation in the presence of xanthohumol. The sterilization method of the present invention can be carried out by performing light irradiation after xanthohumol has been placed in the light irradiation site. The sterilization method of the present invention can be used to assist in dental treatment. The sterilization method of the present invention can be carried out according to the description of the bactericide of the present invention. [Examples]

[0026] The present invention will be described more specifically based on the following examples, but the present invention is not limited to these examples.

[0027] Example 1: Bactericidal effect of photodynamic therapy using xanthohumol The bactericidal effect of Nd:YAG laser was evaluated using xanthohumol.

[0028] (1) Preparation of xanthohumol 217.23g of hop blossoms (Humulus humifusa, Slovenia origin, Otsuya Shoten, Food-sp 246001500) were crushed, extracted with 1 L of a 1:1 mixture of chloroform (CHCl3) and methanol (MeOH), and then concentrated under reduced pressure. This was partitioned between n-hexane (n-Hex) and a mixture of water and methanol (90% aq. MeOH) (1 L each). After concentration, the aq. MeOH layer was partitioned between CHCl3 and water (1 L each), and the CHCl3 layer was concentrated to dryness (32.1622g).

[0029] This CHCl3 layer was repeatedly separated by silica gel column chromatography (mobilization phase: CHCl3-MeOH 1% → 10%, stepwise) to obtain a fraction (1.69 g) that developed yellow color on TLC using phosphomolybdate reagent. This was then processed using Cosmoseal 140 C 18- The fraction (1.2908 g) obtained by separating the sample using OPN reverse column chromatography (Nacalai Tesque) (mobile phase: MeOH 80% → 100%, stepwise) was separated by Toyopal HW-40 fine column chromatography (Tosoh) (mobile phase: CHCl3-MeOH 1:1) to obtain a fraction (615.7 mg).

[0030] Finally, this is Cosmo Seal 140 C 18 - Separation was performed by OPN reverse column chromatography (Nacalai Tesque) (mobile phase: MeOH 70% → 100%, stepwise) to obtain xanthohumol (57.2 mg).

[0031] The sample prepared using the above procedure is dissolved in CD3OH and subjected to nuclear magnetic resonance (JNM-ECZ400S, JEOL) 1 H-NMR and 13 ¹¹C-NMR was measured. From the following measurement results, the substance isolated from hop blossoms was identified as xanthohumol (molecular formula C¹¹C). 21 H 22 It was confirmed to be an O5 compound (see chemical formula below). [ka]

[0032] < 1 H-NMR (CD3OH, 400MHz) δppm> 7.75(1H,d,J=15.6Hz,-CO-CH=C H -),7.62(1H,d,J=15.6Hz,-CO-C H =CH-),7.46(2H,d,J=8.8Hz,φ2,6),6.81(2H,d,J=8.8Hz,φ3,5),5.98(1H,s,φ3'),5.20(1H,m,(CH3)2C=C H -),3.85(3H,s,-OCH3),3.22(2H,d,J=6.8Hz,(CH3)2C=CH-C H 2-),1.75(3H,s,-CH3),1.64(3H,s,-CH3).

[0033] < 13 C-NMR (CD3OH, 100 MHz) δ ppm> 193.2 (C=O), 165.3 (C-4), 162.7 (C-2’), 161.3 (C-4’), 159.9 (C-6’), 142.1 (C-β), 130.5 (C-1), 130.2 (C-2), 129.9 (C-6), 127.5 (C-3”), 124.9 (C-2’), 123.3 (C-5’), 116.1 (C-3), 115.8 (C-5), 108.3 (C-3’), 105.6 (C-1’), 90.6 (C-α), 55.1 (CH3O-), 25.0 (C-5”), 21.3 (C-1”), 16.9 (C-4”).

[0034] (2) Method a Bacterial culture The bacteria to be evaluated was Enterococcus faecalis ( Enterococcus faecalis , which may be referred to as " E. faecalis " in this specification.) (BAA-2128 (trademark), American Type Culture Collection (ATCC)) was used. E. faecalis One colony of 8 was cultured in 10 mL of Brain Heart Infusion (BHI) medium (Sigma-Aldrich) at 37 °C for 24 hours. Centrifugation was performed and the supernatant was discarded, and 3 mL of PBS was added to prepare a bacterial solution. The bacterial solution was placed in 200 μL of PBS and adjusted to 0.2 (OD600) by the McFarland turbidity method, and a bacterial solution of about 2.0×10

[0035] Next, 20 μL of (A) DMSO (control), (B) resveratrol (100 mM) or (C) xanthohumol (100 mM) and 180 μL of BHI solution were added to a 1.5 mL tube (131-715CS, Watoson) and stirred, and about 2.0×10 8 cells of the bacterial solution ( E. faecalis)8 μL was added to prepare the test culture medium. The preparation of test culture media (A), (B), and (C) was performed with n=4. For test substances (B) and (C), DMSO was used as the solvent. The concentration of test substances (B) and (C) in the test culture media was 10 mM.

[0036] I. Laser irradiation For each of the test culture media (A), (B), and (C) obtained in A above, a group was created in which laser irradiation was performed (laser irradiation group) and a group in which laser irradiation was not performed (non-laser irradiation group).

[0037] For the laser-irradiated group, laser irradiation was performed using an Nd:YAG laser (wavelength: 1064 nm, Nd Compact®, Incisive Japan, 140 mJ / pulse, 25 Hz, laser tip 0.4 mm) (output: 3.5 W, continuous wave). Specifically, a 400 μm diameter fiber was used to irradiate a 1.5 mL tube containing bacterial culture medium from a position 6 mm from the bottom for 60 seconds. The non-irradiated group was left undisturbed for 60 seconds without laser irradiation.

[0038] (c) Evaluation of sterilization effect 10 μL was taken from each bacterial culture medium (after laser irradiation and after non-irradiation) and diluted 10-fold with 90 μL of BHI medium, repeating this 10-fold dilution four times (10,000-fold dilution). 50 μL was taken from the last 100 μL and, after being left overnight after laser irradiation, was seeded onto a BHI agar plate (52 g / L distilled water). After incubation at 37°C for 24 hours, the number of colonies that appeared was measured. This experiment was repeated twice to confirm reproducibility (Experiment 1 and Experiment 2).

[0039] (2) Results The results are shown in Table 1 and Figure 1 below. [Table 1]

[0040] In all three groups—control group (A), resveratrol group (B), and xanthohumol group (C)—a significant reduction in bacterial count was observed in the laser-irradiated groups compared to the non-laser-irradiated groups. Furthermore, a significant reduction in bacterial count was confirmed in the laser-irradiated resveratrol group (B) and xanthohumol group (C) compared to the control group (A). In other words, it was confirmed that resveratrol and xanthohumol enhance the bactericidal effect of laser irradiation. Moreover, in the laser-irradiated xanthohumol group (C), a remarkable reduction in bacterial count was observed compared to resveratrol, a type of polyphenol. In other words, it was confirmed with reproducibility that xanthohumol strongly enhances the bactericidal effect of laser irradiation.

[0041] Example 2: Bactericidal preparations containing xanthohumol A mixture of ethanol (95%) and glycerol (volume ratio 1:1) was added to xanthohumol to achieve a xanthohumol concentration of 25 mg / mL (70.5 mM). After dissolving while heating, the mixture was returned to room temperature and drawn into a 1 mL disposable syringe. A root canal needle tip was attached to the syringe tip to create a bactericidal preparation for xanthohumol injection into the tooth root. It was also confirmed that a similar bactericidal preparation could be prepared at a concentration of 35.5 mg / mL (100 mM). These bactericidal preparations did not precipitate during storage at room temperature, nor were precipitates observed at low temperatures (4°C), confirming that they are stable preparations at these concentrations.

Claims

1. A disinfectant containing xanthohumol as the active ingredient, for use in photodynamic therapy.

2. A disinfectant according to claim 1 for enhancing the bactericidal effect of photodynamic therapy.

3. A disinfectant according to claim 1 or 2 for use in dental treatment.

4. The disinfectant according to claim 3, wherein the dental treatment is root canal treatment.

5. A bactericide according to claim 1 or 2 for use in dermatological treatment.

6. The bactericide according to claim 5, wherein the dermatological treatment involves inhibiting the growth of or killing the causative bacteria.

7. The bactericide according to claim 1 or 2, which is applied to the light-irradiated area so that the concentration of xanthohumol is 0.1 mM to 100 mM.

8. A sterilization method using photodynamic therapy, characterized by light irradiation in the presence of xanthohumol (excluding medical procedures on humans).

9. The method according to claim 8, which is a method to assist in dental treatment or dermatological treatment.

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