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We evaluated the SARS-CoV-2-inactivation activity of ozonated glycerol (OG). When a viral solution with 1% fetal bovine serum (FBS) was mixed with test solutions at a ratio of 1:19 and incubated for 20 s, OG with ozone concentrations of over 1000 ppm inactivated ≥ 94.38% of the virus. Extension of the reaction time to 1 h led to the inactivation of ≥ 99.82% of the virus (the viral titer was below the detection limit). Extension to 24 h resulted in concentrations over 200 ppm OG inactivating ≥ 99.87% of the virus (the viral titers were below the detection limit). Next, viral solutions with 1, 20, and 40% FBS were mixed with test solutions at a ratio of 1:19 and incubated for 5 min. Whereas the virucidal activity of 500 ppm OG was very limited in the presence of 1% FBS (79.47% inactivation), it increased in the presence of 20 and 40% FBS (95.13 and 97.95% inactivation, respectively; the viral titers were not below the detection limit). Meanwhile, over 1000 ppm OG inactivated ≥ 99.44% of the virus regardless of the FBS concentration (the viral titers were below the detection limit). Extension of the reaction time to 1 h led to 500 ppm OG inactivating ≥ 99.91 and ≥ 99.95% of the virus with 20 and 40% FBS, respectively (the viral titers were below the detection limit). These results suggested that OG might be useful as a virucidal agent against SARS-CoV-2.

This study aimed to compare the SARS-CoV-2-inactivation activity and virucidal mechanisms of ozonated water (OW) with those of slightly acidic electrolyzed water (SAEW) and 70% ethanol (EtOH). SARS-CoV-2-inactivation activity was evaluated in a virus solution containing 1%, 20% or 40% fetal bovine serum (FBS) with OW, SAEW or EtOH at a virus-to-test solution ratio of 1:9, 1:19 or 1:99 for a reaction time of 20 s. EtOH showed the strongest virucidal activity, followed by SAEW and OW. Even though EtOH potently inactivated the virus despite the 40% FBS concentration, virus inactivation by OW and SAEW decreased in proportion to the increase in FBS concentration. Nevertheless, OW and SAEW showed potent virucidal activity with 40% FBS at a virus-to-test solution ratio of 1:99. Real-time PCR targeting the viral genome revealed that cycle threshold values in the OW and SAEW groups were significantly higher than those in the control group, suggesting that OW and SAEW disrupted the viral genome. Western blotting analysis targeting the recombinant viral spike protein S1 subunit showed a change in the specific band into a ladder upon treatment with OW and SAEW. OW and SAEW may cause conformational changes in the S1 subunit of the SARS-CoV-2 spike protein.

Orento is a traditional Japanese medicinal kampo preparation that is also prescribed in oral care. In oral squamous cell carcinoma cell line CAL27, orento significantly inhibited periodontopathogenic bacterium Porphyromonas gingivalis lipopolysaccharide (LPS) and lipoproteins (PAMP)-stimulated production of interleukin (IL)-6. This suggests that orento negatively regulates PAMP-mediated toll-like receptor (TLR) signaling. Orento significantly suppressed PAMP-stimulated activation of the IL-6 promoter, indicating that orento may suppress the production of IL-6 by PAMP at the transcriptional level. Orento also suppressed TLR-mediated activation of transcription factor nuclear factor-kappa B (NF-kB) that was stimulated by PAMP. This finding indicates that orento may suppress the function and activation of factors involved in TLR signaling, thereby suppressing NF-kB-dependent expression of various genes. Orento suppressed IL-1 receptor-associated kinase (IRAK4), IRAK1, and c-Jun N-terminal kinase (JNK) phosphorylation in PAMP-stimulated CAL27 cells. This result indicates that orento is involved in the initiation of TLR signaling by PAMP and suppresses the downstream signaling pathways of myeloid differentiation primary response gene 88 (MyD88) such as mitogen-activated protein kinase (MAPK) and NF-kB cascades. These findings suggest that orento has an inhibitory effect on the production of inflammatory cytokines.

Background: Orento, a traditional Japanese medicine, is known as Kampo medicine in Japan. We investigated the possible efficacy of Kampo medicine for periodontal disease. In this study, we examined the in vitro effects of orento on the proliferation of the inflammatory cytokines interleukin (IL)-6 and IL-8, the production of type 1 collagen, and the secretion of alkaline phosphatase (ALP) in the human osteosarcoma cell line Saos-2 (Saos-2 cells). Methods: The proliferation of Saos-2 cells was assessed by MTT assay. IL-6 and IL-8 levels, type 1 collagen production and ALP secretion were evaluated using enzyme-linked immunosorbent assay and ALP assays. Saos-2 cells were treated with or without 0.1, 1, 10, 100 and 1000 μg/mL of orento for 24 h. Results: Orento (10 μg/mL) significantly induced the proliferation of Saos-2 cells. At this concentration, orento suppressed IL-6 and IL-8 and enhanced type 1 collagen production and ALP secretion. Conclusions: These results indicate that orento controls the IL-6 and IL-8 secretion and cellular metabolism of osteoblasts, resulting in the secretion of early bone-related biomarkers.

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Ozonated glycerol is glycerol containing ozone, has no unpleasant odor, and has a long half-life. To apply ozonated glycerol for clinical use, ozonated macrogol ointment has been developed by adding macrogol ointment to ozonated glycerol to increase the retention in the affected area. However, the effects of ozone on this macrogol ointment were unclear. The viscosity of the ozonated macrogol ointment was approximately two times higher than that of ozonated glycerol. The effect of the ozonated macrogol ointment on the human osteosarcoma cell line Saos-2 (Saos-2 cells) proliferation, type 1 collagen production, and alkaline phosphatase (ALP) activity were studied. The proliferation of Saos-2 cells was assessed using MTT and DNA synthesis assays. Type 1 collagen production and ALP activity were studied using ELISA and ALP assays. Cells were treated for 24 h with or without 0.05, 0.5, or 5 ppm ozonated macrogol ointment. The 0.5 ppm ozonated macrogol ointment significantly elevated Saos-2 cell proliferation, type 1 collagen production, and ALP activity. These results also showed almost the same trend as for ozonated glycerol.

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