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. 2023 Jul-Aug;37(4):1540-1551.
doi: 10.21873/invivo.13239.

Comprehensive Study of Anti-UVC Activity and Cytotoxicity of Hot-water Soluble Herb Extracts

Maki Izawa  1 , Yusei Otaka  2 , Hiroshi Sakagami  3 , Sei-Ichi Tanuma  3 , Shigeru Amano  3 , Shin Uota  3 , Megumi Inomata  4 , Yuka Kato  5 , Hiroshi Kadokura  5 , Satoshi Yokose  5 , Katsuyoshi Sunaga  6 , Yukari Koga-Ogawa  7 , Giichirou Nakaya  7 , Shinji Kito  2
Affiliations

Comprehensive Study of Anti-UVC Activity and Cytotoxicity of Hot-water Soluble Herb Extracts

Maki Izawa et al. In Vivo. 2023 Jul-Aug.

Abstract

Background/aim: COVID-19 pandemic caused the rapid dissemination of ultraviolet C (UVC) sterilization apparatuses. Prolonged exposure to UVC, however, may exert harmful effects on the human body. The aim of the present study was to comprehensively investigate the anti-UVC activity of a total of 108 hot-water soluble herb extracts, using human dermal fibroblast and melanoma cell lines, for the future development of skin care products.

Materials and methods: Exposure time to UVC was set to 3 min, and cell viability was determined using the MTT assay. Anti-UVC activity was determined using the selective index (SI), a ratio of 50% cytotoxic concentration for unirradiated cells to 50% effective concentration that restored half of the UVC-induced decrease of viability.

Results: Dermal fibroblasts at any population doubling level were more resistant to UVC irradiation than melanoma cells. Both 49 herb extracts recommended by Japan Medical Herb Association (JAMHA) and 59 additional herb extracts showed comparable anti-UVC activity. SI values of selected herbs (Butterbur, Cloves, Curry Tree, Evening Primrose, Rooibos, Stevia, Willow) were several-fold lower than those of vitamin C and vanillin. Their potent anti-UVC activity was maintained for at least 6 h post irradiation, but declined thereafter to the basal level, possibly due to cytotoxic ingredients.

Conclusion: UVC sensitivity may be related to the growth potential of target cells. Removal of cytotoxic ingredients of herb extracts may further potentiate and prolong their anti-UVC activity.

Keywords: UVC sensitivity; dermal fibroblast; malignancy; melanoma; protection; water-soluble herb extract.

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Conflict of interest statement

The Authors confirm that there are no known conflicts of interest associated with this publication and there has been no significant financial support for this work that could have influenced its outcome.

Figures

Figure 1
Figure 1. Experimental procedures of UVC irradiation. The plates with cells were placed at 550 mm distance from the center of a UVC lamp set within a safety cabinet (A). The radiation intensity (measured by radiometer 100 s after UVC lighting, C), was maximum at b and c (B, D). The experimental flow of the determination of anti-UVC activity is shown in E.
Figure 2
Figure 2. Quantification of anti-UVC activity based on CC50 and EC50 values. Human dermal fibroblast, adult HDFa (A) and human COLO679 cells (B) were exposed to UVC irradiation (orange) or not (blue color) for 3 min from a height of 555 mm in the presence of indicated concentrations of cloves. The 50% cytotoxic concentration (CC50) and the concentration that restored half of the UV-induced decrease of viability (EC50) were determined from the dose–response curve. Anti-UVC activity is expressed as the selectivity index (SI=CC50/EC50). Each value represents the mean±S.D. of 3 determinations.
Figure 3
Figure 3. Higher UVC sensitivity of the melanoma cell line over dermal fibroblasts. (Exp. 1) Cell suspensions (100 μl) of young HDFa (17 PDF) (2.9, 1.45, 0.73, 0.39, 0.18, 0.091, 0.045 and 0.023×104 cells/ml), old HDFa (38.5 PDF) (15.4, 7.7, 3.9, 1.9, 0.96, 0.48, 0.24 and 0.12×104 cells/ml), COLO679 (24.4, 12.2, 6.1, 3.1, 1.5, 0.076, 0.38 and 0.19×104 cells/ml) were inoculated in sextuplicate in 96 microwell plates. These cells were incubated for 48 h to allow complete cell attachment. The upper 3 rows were covered with an aluminum foil (to block the UVC penetration). Cells were then exposed to UVC irradiation for 4 or 8 min. After medium change, cells were incubated for 24 or 48 h, and the viable cell number was determined using the MTT method. The viable cell number of UVC-irradiated cells [% of unirradiated control cells (upper 3 rows)] was plotted as a function of the relative cell density [absorbance at 560 nm of unirradiated cells (upper 3 rows)] (Exp. 2). In order to examine the reproducibility of Exp. 1, cells suspension (100 μl) of young HDFa (19PDF) (37, 19, 9.3, 4.6, 2.3, 1.2, 0.58, 0.29×104/ml), old HDFa (40.5PDF) (14, 7, 3.5, 1.8, 0.88, 0.44, 0.22, 0.11×104/ml) and COLO679 cells (79, 40, 20, 9.9, 4.9, 2.5, 1.2, 0.6 × 104/ml) were inoculated in 96 microwell plates. After 48 h, cells were UVC-irradiated for 1, 2, 4 or 8 min, and processed as described in Exp. 1. Each value represents the mean of triplicate determinations.
Figure 4
Figure 4. UVC protective activity of 108 herb extracts (including those recommended by JAMHA) against UVC-irradiated HDFa cells. HDFa cells (16~34 PDL) were irradiated in triplicate for 3 min with 0 (control), 3.9, 7.8, 15.6, 31.3, 62.5, 125, 250, 500, or 1,000 μg/ml of each herb extract in culture medium. After refeeding with fresh culture medium, cells were incubated for 48 h to determine the viable cell number. The SI value was calculated as described in Figure 2. The SI values of all samples are listed in Table II.
Figure 5
Figure 5. UVC protective activity of 108 herb extracts (including those recommended by JAMHA) against UVC-irradiated COLO679 cells. COLO679 cells were exposed to UVC irradiation for 3 min and the SI values were determined as described in Figure 4. The SI values of all samples are listed in Table II.
Figure 6
Figure 6. Comparison of anti-UVC activity between two groups of herb extracts. SI values of JAMHA-recommended herbs (blue) and other herbs (orange) for COLO679 cells (vertical axis) were plotted vs. SI values for HDFa cells (horizontal axis).
Figure 7
Figure 7. Time-lapse study after longer cell culture. HDFa cells (upper panel) and COLO679 cells (lower panel) were UVC irradiated (orange) or not (blue) in triplicate for 3 min, and incubated for 6, 24, or 48 h without medium change, and then refed with fresh medium and incubated for 42, 24, or 0 h (total incubation time kept to 48 h). Viable cell number was then determined using the MTT method. Each value represents the mean±standard deviation of three determinations.
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