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, malignancy, dermal fibroblast, melanoma, water-soluble herb extract, protection
SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) infection caused the coronavirus disease of 2019 (COVID-19) pandemic, accompanied by serious morbidity and mortality (1). This triggered the rapid dissemination of various types of ultraviolet C (UVC) sterilization apparatuses in hospital emergency department waiting rooms, clinics, nursing homes, prison common areas, public libraries (providing the sterilization box for cleaning books), schools, restaurants, and bedrooms (2). While UVC has strong bactericidal (3) and virucidal (4) activity, the prolonged exposure to UVC may have harmful effects on the human body, causing various diseases such as skin cancer (5) and cataracts (6). Therefore, the search for nontoxic UVC protective substances is crucial. Sakagami et al. recently reported that (i) human normal cells (gingival, periodontal, and dermal fibroblasts and pulp cells] are generally resistant to UVC irradiation, as compared with human cancer cell lines (oral squamous cell carcinoma, lung cancer, glioblastoma, leukemia, and melanoma) (7); and that (ii) phenylpropanoids, which are degradation products of lignin, showed comparable anti-UVC activity with sodium ascorbate (8). The phenylpropanoid biosynthetic pathway provides flavonoids that exert UV protective effects (9) and are used as plant-derived medicinal ingredients (10).
Herbs are used for cooking, maintenance, and improvement of health in daily life. The Japan Medical Herb Association (JAMHA) (11) has engaged in disseminating useful information of their medicinal efficacy. In the present study, we comprehensively investigated the anti-UVC activity of a total of 108 hot-water soluble herb extracts (including 49 herb extracts recommended by JAMHA) against human dermal fibroblast (HDFa) and melanoma (COLO679) cell lines. For future exploration of their application as skin care products, it was necessary to investigate the cytotoxicity and the anti-UVC activity of the herbs. Since our newly established anti-UVC assay system uses culture medium [DMEM supplemented with 10% heat-inactivated fetal bovine serum (FBS)] instead of phosphate-buffered saline without calcium and magnesium (PBS) [that causes cell detachment and damage during irradiation (7)], we could incubate the cells for much longer time for the detection of cytotoxicity (7) (Method II, Figure 1E). Using this new method, we also investigated to which extent the anti-UVC activity of selected herb extracts is maintained after prolonged incubation time.
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.
Materials and Methods
Materials. Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Phosphate-buffered saline without calcium and magnesium (PBS) was obtained from Nissui Pharmaceutical Co. (Tokyo, Japan). Dimethyl sulfoxide (DMSO) was from Wako Pure Chemical Ind. (Osaka, Japan). 96-microwell plates were from Techno Plastic Products AG (Trasadingen, Switzerland).
Preparation of herb extracts. Herbs (1 g) were extracted for 30 min with 20 ml of pure water (Milli-Q) at 100˚C. After cooling down, the hot-water extract was filtered and then freeze-dried. A total of 108 hot-water soluble herb extracts [including 49 herb extracts recommended by JAMHA] are listed in Table I.
Table I. Lists of 108 herb extracts used in the present study.
Cell culture. Human dermal fibroblasts, adult (HDFa) isolated from adult skin and cryopreserved at the end of the primary culture (catalog number: C0135C; Thermo Fisher Scientific, Waltham, MA, USA) and human melanoma cells (COLO679) (catalog number: R21-0267; Riken Cell Bank, Tsukuba, Japan) were cultured at 37˚C in regular culture medium [DMEM supplemented with 10% heat (56˚C, 30 min)-inactivated FBS, 100 U/ml penicillin G, and 100 μg/ml streptomycin sulfate] in a humidified incubator (MCO-170 AICUVD-P; Panasonic Healthcare Co., Ltd., Gunma, Japan) with 5% CO2 as described previously (7). For the subculture, HDFa cells were harvested using 0.25% trypsin-EDTA and seeded at a 1:4 ratio once a week, with an intermediate medium change after 4 days, causing the increase in the population doubling level (PDL) by two for one week. HDFa cells were used at 16-34 PDL in this study.
UVC irradiation condition. The 96-microwell plates (on which cells were growing) were placed at 550 mm distance from the center of a UVC lamp (254 nm, germicidal lamp GL15; Toshiba Co., Tokyo, Japan) set within an MCV-B131F BioClean Bench chamber (Panasonic Healthcare Co., Tokyo, Japan) (A in Figure 1). The radiation intensity in the center below the UVC lamp (point b in B) measured using a UVC radiometer (Gigahertz Optik GmbH, Tuerkenfeld, Germany) was 1.193 W/m2 (C), comparable with that at point c, but larger than that at point a (D). Based on these data, the 96-microwell plates were placed in the center (point b) for UVC irradiation (Figure 1).
UVC protection assay. Cells were inoculated at the indicated cell density in the inner 60 wells of a 96-microwell plate. The surrounding 36 exterior wells were filled with 150 μl of sterile distilled water to minimize the evaporation of water from the culture medium (Figure 1A). Cells were incubated for 48 h to complete attachment to the plate. After medium change with fresh culture medium containing various concentrations of test samples, cells were irradiated with UVC as described above. After the medium change (method I) or without medium change (method II) (Figure 1E), cells were incubated for various times (0 to 48 h) and their viability was determined using the MTT method (7,8). Briefly, the treated cells were incubated for another 2 h in fresh culture medium containing 0.2 mg/ml MTT. Cells were then lysed with 0.1 ml DMSO and the absorbance at 560 nm of the cell lysate was determined using a microplate reader (Infinite F 50 R, TECAN, Kawasaki, Japan). The 50% cytotoxic concentration (CC50) and the concentration that abolished the UVC-induced loss of viability by 50% (EC50) were determined in triplicate from the dose-response curve. The selectivity index of UVC protection (SI) was determined using the following equation: SI=CC50/EC50, as described in 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.
Statistical analysis. Each experimental value is expressed as the mean±standard deviation (SD) of triplicate determinations. Student t-test was performed for statistical analysis of differences between two groups (paired and unpaired). The significance level was set at p<0.05.
Results
Comparison of UVC sensitivity between young and old HDFa, and COLO679. Young and old human dermal fibroblasts (HDFa) (17PDL and 38.5PDL, respectively) and COLO679 cells were compared for their UVC sensitivity. Cell suspensions (0.1 ml) at various densities were inoculated into a 96-microwell plate and incubated for 48 h in culture medium (DMEM supplemented with 10% heat-inactivated FBS) to allow complete cell attachment. After refeeding with fresh culture medium, the cells were exposed to UVC irradiation for 4 or 8 min. After incubation for 48 h in fresh culture medium, the viable cell number was determined using MTT method (Method I in Figure 1E). Then, the viable cell number of UVC-irradiated cells (% of control) was plotted as a function of cell density [expressed as A560 (absorbance at 560 nm) of unirradiated cells]. When the exposure time to UVC was increased from 4 to 8 min, viable cell number declined. Similarly, prolongation of incubation time from 24 to 48 h also reduced the viable cell number. In all cases, COLO679 were the most sensitive to UVC irradiation, followed by young HDFa, and then old HDFa, under the condition that the cell density (assessed by A560) was between 0.1 to 1 (Exp. 1, Figure 3). Reproducibility of these findings was confirmed (Exp. 2, Figure 3). The viable cell number declined with an increase in the UVC irradiation time (from 1 to 8 min) and incubation time (from 24 to 48 h). COLO679 cells again showed the highest UVC sensitivity, followed by young and then old HDFa cells, suggesting the relation of UVC sensitivity to the growth potential.
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.
Search for UVC protective herb extracts (with Method I). Relative anti-UVC activity of 108 hot-water soluble herbs was investigated using HDFa (Figure 4) and COLO679 cells (Figure 5), and Method I (Figure 1E). The CC50, EC50 and SI values were determined from the dose-response curves (Table II). Among 49 herb extracts recommended by JAMHA, cloves [SI=17.1 (HDFa); 9.4 (COLO679)], evening primrose (SI=12.7; 8.0), and thyme (SI=12.4; 4.4) showed higher anti-UVC activity. Among the 59 other herbs, butterbur (SI=12.8; 5.3), curry tree (SI=15.9; 3.3), rooibos (SI=13.3; 6.0), stevia (SI=13.6; 6.7) and willow (SI=12.4; 5.5) showed higher anti-UVC activity. When the SI values of a total 108 extracts [49 JAMHA-recommended herb extracts (blue color) and other 59 herb extracts (red color)] for HDFa (x-axis) and COLO679 cells (y-axis) were plotted, two regression almost superimposed lines were produced (JAMHA recommended herbs: y=0.5729x, R2=0.91; other herbs: 0.4804x, R2=0.9004) (Figure 6). JAMHA recommended herbs (n=49) produced significantly higher anti-UVC activity in HDFa cells (SI=4.29±3.64) than in COLO679 cells (SI=2.73±2.00) (p<0.01), possibly due to higher background level that is high % of viability following UV irradiation without sample, which reflects higher UVC resistance in the former cells. Similarly, the other herbs (n=59) showed higher anti-UVC activity in HDFa cells (SI=4.19±3.64) than in COLO679 cells (SI=2.41±1.44) (p<0.001). These two groups of herb extracts showed indistinguishable anti-UVC activity on HDFa (p=0.88) and COLO679 (p=0.34) cells.
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. 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.
Table II. Anti-UVC activity of two groups of medical herb extracts.
Bold values represent medical herb extracts that showed higher anti-UVC activity.
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).
Evaluation of Stability of anti-UVC activity (with Method II). Finally, the stability of the anti-UVC activity of the most potent herb extracts (evening primrose, rooibos, and stevia) during long culture, was evaluated using DMSO as solvent control. HDFa cells were irradiated for 3 min in the presence of various concentrations of these herbs, and incubated for a further 6 h, followed by medium change and incubation for 18 h with fresh culture medium (Method II, Figure 1E). Evening primrose and stevia maintained high anti-UVC activity (SI=12.8 and 11.2, respectively). We confirmed that incubation with 1% DMSO for 6 h did not affect the viability of both HDFa and, COLO679 cells (rightmost column, Figure 7). Longer incubation for 24 and 48 h without medium change resulted in the complete disappearance of anti-UVC activity (SL=1.0~3.1), possibly due to the cytotoxicity of DMSO, or cytotoxic ingredients since these extracts are mixtures of active and other biologically inert or antagonistic components (upper panel, Figure 7). Similarly, rooibos and stevia maintained significant anti-UVC activity (SI=4.2, 3.0) for 6 h in COLO679 cells (lower column, 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.
Discussion
The present study investigated for the first time the anti-UVC activity of a total of 108 medicinal herb extracts. First, we demonstrated that the mean SI value (anti-UVC activity) of 49 herb extracts recommended by JAMHA was comparable to that of other 59 herb extracts: 4.29±3.64 vs. 4.19±3.64 (p=0.88) for HDFa cells; 2.73±2.00 vs. 2.41±1.44 (p=0.33) for COLO679 cells. This indicates that not only the JAMHA recommended herbs, but also other herbs can be utilized for exploring skin protective products.
Among them, eight species (butterbur, cloves, curry tree, evening primrose, rooibos, stevia, willow) showed higher anti-UVC activity. Their anti-UVC activity (SI=12.4~17.1 for HDFa cells and SI=3.3~9.4 for COLO679 cells) were nearly 1/5~1/10 that of vitamin C and lignin degradation products (vanillin, vanillic acid, p-coumaric acid, caffeic acid, ferulic acid, chlorogenic acid) (SI=67~321 for HDFa cells and SI=25~79 for COLO679 cells) (8). Evening primrose and stevia maintained high anti-UVC activity at least for 6 h after UVC irradiation. The presence of 1% DMSO (a solvent used for dissolving the samples) for 6 h did not affect cell viability. Therefore, the experimental data presented in Figure 3, Figure 4, Figure 5, and Figure 6 removing herbs just after 3 min UVC irradiation were not affected by the solvent. We found that following incubation with herbs for more than 6 h the SI values were rapidly declined, possibly due to cytotoxic components of the herb extracts or DMSO. At present, the major influencing factor is not clear. It is preferable to use noncytotoxic herb extracts for longer treatment. The removal of cytotoxic substance by organic solvent extraction may elevate and maintain for longer time their UVC activity, as we have experienced for the enhancement of antimicrobial activity of mastic following removal of cytotoxic ingredients using n-hexane extraction (12). The present assay system using culture medium instead of PBS may be useful to search for non-cytotoxic herb species.
We found that COLO679 cells showed much higher sensitivity, followed by young HDFa and then old HDFa, confirming our previous finding (7). This suggests that UVC sensitivity of cells may be tightly coupled to their growth potential. The tumor-specific cytotoxic effects of herb extracts may synergistically strengthen the anticancer activity of antineoplastic agents (13-15).
Rooibos extracts and their components such as aspalathin, nothofagin, vitexin, isovitexin, and eriodictyol, as well as the structurally related phloretin and phloroglucinol have been reported to exhibit the strongest methylglyoxal-trapping and antiglycative activities against advanced glycation end product formation (16,17). Rooibos extracts also reduced reactive oxygen species and stimulated the catalase and superoxide dismutase enzyme activities (18). It has been also reported that Oenothera biennis (evening primrose) induced a small but significant reactivation of senescent human dermal fibroblasts (19). It also showed antioxidative and anti-inflammatory actions in diet-induced obese mice (20). Likewise, Stevia rebaudiana extract rich in phenols and flavonoids possessed significant antioxidant activities and a promising whitening effect, having potential use as active ingredients in cosmetic products (21).
The anti-UVC activity of herb extracts is affected by many factors: antioxidant activity (such as hydroxyl radical scavenging activity), repair system (reduction of DNA lesions in the nuclei of cells exposed to UVC) (22) and the presence of cytotoxic components. Since phenylpropanoids scavenge hydroxyl radicals more efficiently than superoxide radical, 1,1-diphenyl-2-picrylhydrazyl radical and 2,2’-zzinobis-3- ethylbenzothiazoline-6-sulfonic acid radical (23), their anti-UVC activity at the initially stage (3 min irradiation) may be mostly affected by their antioxidant action. Since evening primrose, and stevia showed comparable anti-UVC activity with rooibos (Figure 4, Figure 5, and Figure 7), fractionation of their active components is underway.
Conflicts of Interest
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.
Authors’ Contributions
MI, YO, and HS performed the experiments. HS wrote the article. S-IT, SA, SU, MI, YK, HK, SY, KS, YK-O, GN, and SK reviewed the article. HS interpreted the experimental results and edited the article. All Authors read and approved the final version of the article.
Acknowledgements
The Authors thank Mr. Koichiro Yano for his technical support. This work was supported by Miyata Research Fund B.
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