Abstract
Background/Aim: The rapid spread of COVID-19 resulted in the revision of the value of ultraviolet C (UVC) sterilization in working spaces. This study aimed at re-evaluating the anti-UVC activity of four groups of natural products against human melanoma COLO679 and human normal dermal fibroblast (HDFa) cells, based on chemotherapeutic index. Materials and Methods: Various cell lines were exposed to UVC for 3 min in the presence of increasing concentrations of test compounds and viable cell numbers were determined with the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. The anti-UVC activity was quantified by the ratio of the 50% cytotoxic concentration (determined without irradiation) to the 50% effective concentration (which abolished by 50% the UVC-induced loss of viability). Apoptosis was quantified as the subG1 population proportion following cell-cycle analysis. Results: Among four groups of major natural products, six phenylpropanoids showed the highest anti-UVC activity, followed by the lignified products and alkaline products that contain lignin and its degradation products. On the other hand, tannins and flavonoids showed lower activity due to their higher cytotoxicity. UVC-sensitive COLO679 cells lack dectin-1 protein expression. Conclusion: These data suggest the prominent anti-UVC activity of lignin degradation products, and the possible involvement of dectin-1 expression in UVC-sensitivity.
Since the identification of a novel coronavirus (severe acute respiratory syndrome coronavirus 2, SARS-CoV-2), coronavirus disease 2019 (COVID-19) has spread worldwide (1). Coronavirus infects the transbronchial and alveolar epithelial cells, inducing lung damage and other organ impairments (2), and long-lasting spread of infection aggravates psychological and physical complications (3, 4). Accordingly, the importance of environmental cleaning with sodium hypochlorite, disinfection with bactericidal and viricidal ultraviolet C (UVC) lamps in contaminated rooms and wearing masks has been designated as effective methods to reduce the risk of infection (5, 6).
Moderate doses of UV exert several favorable effects such as induction of vitamin D biosynthesis (7), hormetic response in plant tissues (8, 9), and increasing reproductive performance in Tigriopus californicus (10). However, increased time spent in rooms with a UVC lamp may gradually produce harmful effects. Actually, excessive UVC radiation absorption by the epidermis produces reactive oxygen species, and causes various cutaneous disorders such as photoaging and skin cancer (11). Since UVC sterilization lamps, now available through internet shopping, have been introduced for home usage, it is necessary to investigate measures protecting from UVC injury.
Due to the increased distribution of UVC equipment, the exploration of safe and effective UVC-protective substances is crucial. Most previous studies have investigated small numbers of compounds, without presenting a chemotherapy index (reflecting both cytotoxicity and efficacy). In the present study, we compared the anti-UVC activity of several groups of natural products to clarify which chemical structures are essential for exerting potent anti-UVC activity, using UVC-resistant human dermal fibroblast (HDFa) and UVC-sensitive (COLO679) cells (12).
We reported the prominent anti-UVC activity of lignin–carbohydrate complex (LCC) (13). LCC from Lentinus edodes mycelia extract enhanced gene expression of dectin-2, a C-type lectin receptor (14) which recognizes microbial polysaccharides (15). This suggests a significant role of the activation of the dectin-2 signaling pathway as a result of LCC action. Since dectin-2 is involved in tolerance of UV radiation (16, 17), the question arose whether UVC-resistant HDFa cells express higher amounts of dectin-2 protein than UVC-sensitive COLO679 cells. However, contrary to our expectation, we found both cell lines expressed comparable amounts of dectin-2 protein (12). A recent study suggested that dectin-1, another C-type lectin receptor (14), has a role distinct from that of dectin-2 in skin wound-healing through its different effects on neutrophilic inflammatory response (18). Therefore, in order to clarify the possible role of dectin-1 in UVC sensitivity, dectin-1 protein expression was compared in UVC-sensitive and -resistant cells.
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 Inc. (St. Louis, MO, USA). Phosphate-buffered saline without calcium and magnesium (PBS) were purchased from Nissui Pharmaceutical Co. Ltd., (Tokyo, Japan). (−)-Epigallocatechin-3-gallate (EGCG), dimethyl sulfoxide (DMSO) and catalase (from bovine liver, 8,000 U/ml) were purchased from Fuji Film Wako Pure Chemical Industry (Osaka, Japan). Sodium ascorbate, vanillin, vanillic acid, gallic acid, p-coumaric acid, caffeic acid, trans-ferulic acid, isoferulic acid (hydroxycinnamic acid), chlorogenic acid, alkali-lignin, humic acid, and N-acetyl-L-cysteine (NAC) were purchased from Tokyo Chemical Industry Co., Ltd., (Tokyo, Japan). Lignosulfonate B (MW 33600, purity: 92%) (19) was supplied from Nippon Paper Industries Co., Ltd., (Tokyo, Japan). Alkaline extract of the leaves of Sasa sp. (SE) (20) was provided by Daiwa Biological Research Institute Co., Ltd. (Tokyo, Japan). Lastly, 96-microwell plates were from Techno Plastic Products AG (Trasadingen, Switzerland).
Cell culture. Human dermal fibroblast adult cells (HDFa, used at 20-30 population doubling) and human melanoma cells (COLO679) (Riken Cell Bank, Tsukuba, Japan) were cultured at 37°C in 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 (12).
UVC protection assay. Cells were inoculated at 3×103 cells/0.1 ml 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 evaporation of water from the culture medium. After incubation for 48 h, the medium was replaced by fresh culture medium containing different concentrations of the following compounds: sodium ascorbate (used as positive control), vanillin, vanillic acid, p-coumaric acid, caffeic acid, trans-ferulic acid, isoferulic acid, chlorogenic acid, alkali-lignin, lignosulfonate B, SE, pine seed shell extract, humic acid, tannic acid, EGCG or gallic acid, resveratrol (generally 0, 7.8, 15.6, 31.3, 62.5, 125, 250, 500, 1,000 or 2,000 μg/ml); NAC (0, 15.6, 31.3, 62.5, 125, 250, 500, 1,000, 2,000 or 4,000 μg/ml) or catalase (0, 39, 78, 156, 313, 625, 1,250, 2,500, 5,000 or 10,000 units/ml). For stock solutions, resveratrol was dissolved in DMSO at 25 mg/ml while other compounds were dissolved directly in culture medium at 2 mg/ml, and then sterilized by passing through a Millipore filer (0.45 μm, Merck Millipore Ltd., County Cork, Ireland). Humic acid solution was not passed through the Millipore filter due to its water-insolubility. The plates were then placed at 550 mm from the center of a UV lamp (germicidal lamp GL15; Toshiba Co. Ltd., Tokyo, Japan) set within a safety cabinet (MCV-B131F BioClean Bench; Panasonic Healthcare Co., Ltd., Tokyo, Japan). The culture plate was placed in the corner of the cabinet against the steel wall to absorb the vertically applied UVC light (Figure 1A). The detailed procedures are described in our recently published paper (12). Irradiation power was determined by a UVC radiometer (Gigahertz Optik GmbH, Tuerkenfeld, Germany). Cells were then irradiated at 1.022 W/m2 for 3 min unless otherwise stated, then refed with fresh culture medium, and incubated for a further 48 h. Cell viability was subsequently determined by the MTT method (Figure 1B). From the dose-response curve, the 50% cytotoxic concentration (CC50) and the concentration that abolished by 50% the UV-induced loss of viability (EC50) were determined in triplicate (Figure 2). The selectivity index of UVC protection (SI) was determined using the following equation: SI=CC50/EC50. The DMSO concentration in the resveratrol-treated cell culture was below 1%. The cytotoxicity of DMSO at each concentration of resveratrol (0.0313, 0.0625, 0.125, 0.25, 0.5, 1%) alone was subtracted.
Experimental procedures of UVC irradiation (A), and assays for cytotoxicity using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (B), cell-cycle distribution (C) and dectin-1 protein expression (D), using cell sorting. HDFa and COLO679 cells were inoculated in the inner 60 wells of 96-microwell plates to determine the viable cell number (B), or in a 10-cm dish to determine the distribution of cells in subG1, G1, S and G2/M phases (C) and dectin-1 protein expression (D).
Method for determination of anti-UVC activity. Human dermal fibroblast HDFa (A) and human COLO679 cells (B) were inoculated into 24 plates (two plates for each sample). Twelve plates were exposed to UVC irradiation for 3 min from a height of 555 mm in the presence of the indicated concentration of lignosufonate B; the other 12 plates were not irradiated. From the dose-response curve, the 50% cytotoxic concentration (CC50) and the concentration that abolished by 50% the UV-induced loss of viability (EC50) were determined. Anti-UVC activity was expressed as the selectivity index of UVC protection (SI=CC50/EC50). Each value represents the mean±S.D. of three determinations.
Cell sorter analysis. Apoptosis induction was evaluated by cell-cycle analysis as described previously (21). Briefly, cells were treated for 24 h without (control), actinomycin D (1 μM), lignosulfonate B (500 μg/ml) or vanillin (250 μg/ml). Cells were then fixed, digested with RNase A, stained with propidium iodide, filtered through Falcon® 40 μm cell strainer (Corning, Inc., Corning, NY, USA) and then subjected to cell sorting (Figure 1C).
Dectin-1 protein expression was evaluated by flow cytometry, under a similar set up previously described for dectin-2 (12). Briefly, the cells (1×106 cells/well) were stained with rabbit monoclonal antibody against human dectin-1 (E1X3Z) (Cell Signaling Technology Inc., Danvers, MA, USA) at room temperature for 1 h. After washing with PBS, the cells were incubated with Alexa488-conjugated anti-mouse IgG antibody (Thermo Fisher Scientific, Waltham, MA, USA) at room temperature for 1 h. The samples were analyzed using an SH800 cell sorter (Sony, Tokyo, Japan) (Figure 1D).
Statistical analysis. Each experimental value is expressed as the mean±standard deviation of triplicate determinations. One-way analysis of variance and Bonferroni’s post-hoc test were performed using IBM SPSS 27.0 (IBM Corp., Armonk, NY, USA). The significance level was set at p<0.05.
Results
Optimal conditions of UVC irradiation. We first described how to quantitate the anti-UVC activity, using 3-min irradiation time [most favorable exposure time (12)]. The power of UVC reaching the bottom of the safety cabinet (555 mm from the UVC lamp) was 1.022 W/m2 (12). When HDFa and COLO669 cells were exposed to UVC irradiation for 3 min, followed by incubation for 24 h in fresh DMEM with 10% FBS, their viability declined by 55.1 and 97.3%, respectively, confirming the higher UVC sensitivity of COLO679 cells (Figure 2). In the presence of increasing concentrations of lignosulfonate B, the viable cell number of UVC-irradiated cells gradually increased. According to the dose-response curve using 3 min-irradiation time, the EC50 was 184 and 278 μg/ml for HDFa and COLO669 cells, respectively. Since the CC50 was more than 1,000 μg/ml for both cell lines, SI values were determined to be >5.4 and >3.6, respectively (Figure 2).
Using this quantification method, we next determined the optimal irradiation time. When the exposure time was 2 min or less, viability of HDFa cells declined by 50% or less. Prolongation of irradiation time to 3, 5, 7 or 10 min reduced the viability to a plateau: 34.9%, 30.9%, 32.6% and 38.7% of the control, respectively, in HDFa cells (Figure 3A) and 7.8, 3.7%, 3.5% and 2.7% of the control, respectively, in COLO679 cells (Figure 3B). An irradiation time of 3 min produced a higher SI value for lignosulfonate B than those attained by irradiation time of 5, 7 or 10 min [>14.5 vs. >11.0, >10.2 and >9.0, respectively, with in HDFa cells (Figure 3A) and >10.9 vs. >9.3, >9.2 and >7.6, respectively, in COLO679 cells (Figure 3B)]. Based on these finding, the experiments that followed adopted the condition of 3-min exposure to UVC irradiation.
Determination of optimal UVC irradiation time. HDFa (A) and COLO679 cells (B) were exposed to UVC irradiation for 0, 1, 2, 3, 5, 7 or 10 min in the presence of the indicated concentrations of lignosulfonate B. The medium was then removed, replaced with fresh Dulbecco’s modified Eagle’s medium with 10% fetal bovine serum and cells were incubated for 48 h then the viable cell number was determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide method. The anti-UVC activity was expressed as the selectivity index (SI) for cells exposed to UVC for 3 min or more: SI=50% cytotoxic concentration (CC50)/concentration that abolished by 50% the UV-induced loss of viability (EC50). Each value represents the mean±S.D. of three determinations. Note that the concentration axis is exponential.
Effect of filtration and type of medium on anti-UVC activity. Figure 4 shows that the anti-UVC activity of lignosulfonate B did not decline after filtration but, for unknown reasons, slightly increased (SI>26.5). Regardless of filtration, a higher SI value was observed when cells were irradiated in culture medium (DMEM with 10% FBS) than in PBS (Figure 4). This suggests that UVC irradiation in PBS might have produced more detached or damaged cells, compared to culture medium, supporting our previous finding (12). Based on this, the experiments that followed were performed using culture medium under UVC irradiation.
Anti-UVC activity of lignosulfonate B was not reduced by passing through a Millipore filter. Lignosulfonate B was dissolved in Dulbecco’s modified Eagle’s medium (DMEM) plus 10% fetal bovine serum (FBS) or phosphate-buffered saline (PBS) at 2,000 μg/ml, filtered through a Millipore filter (cut-off: 0.45 μm) or not. HDFa cells were exposed for 3 min in DMEM/10%FBS or PBS with the indicated concentrations of filtered or unfiltered lignosulfonate B. Cells were then incubated for 48 in fresh culture medium to determine the viable cell number. Each value represents the mean±S.D. (n=3). Note that the concentration axis is exponential.
Comparison of apoptosis induction and dectin-1 levels between UVC-sensitive and -resistant cells. Figure 5 shows that UVC irradiation induced an accumulation of the subG1 population (indicator of DNA fragmentation) in both HDFa and COLO679 cells. The percentage of the subG1 population was significantly higher in COLO679 cells compared to HDFa cells (18.8% vs. 3.3% at 13-14 h after irradiation; p<0.05); and (29.2% vs. 7.0% at 24 h after irradiation; p<0.05). The increase of the subG1 population paralleled the reduction of G2/M phase cells (Figure 5B). This suggests the occurrence of DNA fragmentation following accumulation of cells in the G2/M phase.
UVC-sensitive cells produced a greater population of subG1 cells upon irradiation than did UVC-resistant cells. HDFa (A) and COLO679 (B) cells were exposed to UVC irradiation for 3 min, and then incubated for 14 (or 13) or 24 h before cell-cycle analysis. Significantly different at: *p<0.05 vs. control; *p<0.05 [Lignosulfonate B (+) vs. Lignosulfonate B(−)]; *p<0.05 [vanillin (+) vs. vanillin (−)]
Cell surface expression of dectin-1 was investigated in both HDFa and COLO679 cells. Surprisingly, dectin-1 protein was detected only in HDFa cells, and not in COLO679 cells (Figure 6). This finding is quite different from dectin-2 expression, which was observed in both of these cell lines (12). Absence of dectin-1 protein in COLO679 cells may explain, at least in part, their higher susceptibility to UVC irradiation.
Expression of dectin-1 in unirradiated HDFa (A) and COLO679 (B) cells quantified by flow cytometry. Red: Stained with dectin-1 antibody; Green: stained with control antibody; Blue: unstained.
Anti-UVC activity of phenylpropanoids. The anti-UVC activity of four groups of natural products was investigated using sodium ascorbate as a positive control (12). Representative dose-response curves of cytotoxicity without and with UVC irradiation are shown in Figure 7, and Figure 8. The CC50 and EC50 values are summarized in Table I. All compounds and extracts showed 1.0- to 8.7-fold higher anti-UVC activity when HDFa cells were used, compared to COLO679 cells, possibly corresponding to higher numbers of viable cells after UVC irradiation.
Dose–response curves of the anti-UVC activity of natural products against HDFa and COLO679. HGFa (A, B) and COLO679 (C) cells were irradiated or not for 3 min in Dulbecco’s modified Eagle’s medium with 10% fetal bovine serum containing the indicated concentrations of test compounds, and then incubated for 48 h with fresh culture medium. Viable cell number was determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide method, and data were plotted as viable cell number (% of unirradiated control). From the dose–response curve, the 50% cytotoxic concentration (CC50) and the concentration that abolished by 50% the UVC-induced loss of viability (EC50) were determined. The selectivity index (SI) was determined by the following equation: SI=CC50/E50. Each value represents mean±S.D. of triplicate determinations. EGCG: (−)-Epigallocatechin-3-gallate; SE: alkaline extract of the leaves of Sasa sp.; NAC N-acetyl-L-cysteine. Note that the concentration axis is exponential.
Anti-UVC activity of four groups of natural products.
Group I compounds included phenylpropanoids (components of lignin) and related compounds. Vanillin, vanillic acid, p-coumaric acid, trans-ferulic acid, isoferulic acid (data of only one experiment), and chlorogenic acid showed the highest anti-UVC activity (SI >67-321) among the four groups, comparable to that of sodium ascorbate, used as a positive control (SI>135) (12).
Group II included lignified materials (alkali-lignin, lignosulfonate B) and alkaline plant extracts of the leaves of Sasa sp (SE) and pine seed shell. These substances had slightly lower anti-UVC activity (SI >12-48) compared to Group I. Humic acid is an amorphous material formed by the microbial degradation of lignin and charcoal, is widespread in soils, rivers, oceans, and coal-related natural resources (22). It is only soluble in alkaline solution, not in water, and therefore showed no anti-UVC activity (Table I). It should be noted that lignin degradation products (Group I) show approximately four times higher anti-UVC activity (mean SI>135) than that of authentic lignified materials (Group II) (mean SI>34) (Table I).
Group III included tannin-related compounds. Tannic acid, epigallocatechin gallate (a major component of green tea) and gallic acid (component unit of tannin) showed anti-UVC activity to comparable extent attained by Group II substances (SI>18-43).
On the other hand, Group IV (antioxidants), such as resveratrol (a flavonoid), N-acetyl-L-cysteine (glutathione donor), and catalase (which degrades hydrogen peroxide) showed essentially no anti-UVC activity at concentrations tested (Figure 7A and Figure 8; Table I).
Dose-response curves of anti-UVC activity of natural products and catalase in HDFa cells. HDFa cells were irradiated or not for 3 min in Dulbecco’s modified Eagle’s medium with 10% fetal bovine serum containing the indicated concentrations of test compounds, and then incubated for 48 h with fresh culture medium. Viable cell number was determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide method, and data were plotted as viable cell number (% of unirradiated control). From the dose–response curve, the 50% cytotoxic concentration (CC50) and the concentration that abolished by 50% the UV-induced loss of viability (EC50) were determined. The selectivity index (SI) was determined by the following equation: SI=CC50/E50. EGCG: (−)-Epigallocatechin-3-gallate; SE: alkaline extract of the leaves of Sasa sp.; NAC N-acetyl-L-cysteine. Note that the concentration axis is exponential.
Discussion
The present study compared the anti-UVC activity of lignin (alkali-lignin, lignosulphonate B) (Group II) and its degradation products such as phenylpropanoids (p-coumaric acid, caffeic acid, trans-ferulic acid, isoferulic acid) and structurally-related compounds (vanillin, vanillic acid, chlorogenic acid) (23, 24) (Group I). We found for the first time that lignin degradation products (Group I) show approximately four times higher anti-UVC activity than that of authentic lignified materials (Group II) (Table I). The prominent anti-UVC activity of Group I did not depend on the presence (caffeic acid and chlorogenic acid) or absence (vanillin, vanillic acid, p-coumaric acid, trans-ferulic acid, isoferulic acid) of a catechol group. On the other hand, flavonoids such as resveratrol (Group III) had much lower anti-UVC activity (SI<0.3), possibly due to their higher cytotoxicity (lower CC50 values) than Group I (indicated by blue lines in Figure 7A and B, respectively). It should be noted that gallic acid showed very potent cytotoxicity (Figure 8).
There are at least three possible mechanisms of anti-UVC activity of phenylpropanoids: (i) Scavenging of hydroxyl radical, (ii) supplementation of intracellular glutathione, and (iii) repair of cell damage caused by UVC irradiation, or hormetic growth stimulation. The following evidence supports the first mechanism: Tumor cells produce higher amounts of hydrogen peroxide than normal cells (25), and hydrogen peroxide is rapidly converted into hydroxyl radical by UV from an ultraviolet lamp (26). Mathew et al. reported that phenylpropanoids scavenge hydroxyl radical more efficiently than superoxide radical, 1,1-diphenyl-2-picrylhydrazyl radical and 2,2’-azinobis-3- ethylbenzothiazoline-6-sulfonic acid radical, and gallic acid, a structural unit of tannin, showed slightly higher hydroxyl radical-scavenging activity than vanillin and vanillic acid (17). The order of inhibition of hydroxyl radical-scavenging activity measured by the deoxyribose method in a cell-free system (27) was a slightly different from the anti-UVC activity measured by cell culture system in the present study where the anti-UVC activity of gallic acid was slightly lower than that of phenylpropanoids such as vanillin and vanillic acid (Table I). This discrepancy might be due to the higher cytotoxicity of gallic acid (blue line in Figure 8). Catalase, which decomposes hydrogen peroxide, failed to show any detectable anti-UVC activity (Table I). This suggests the importance of intracellular hydrogen peroxide, which cannot be removed by exogenously added catalase.
On the other hand, the second proposed mechanism, namely the involvement of glutathione, may not be likely, since N-acetyl-L-cysteine, an agent that increases the intracellular concentration of glutathione (28), failed to prevent UVC-induced cell injury (Figure 7A and Figure 8).
As for the third mechanism, in other studies, we found that SE partially alleviated doxorubicin-induced keratinocyte cytotoxicity (29), and inhibited amyloid β-induced cytotoxicity against both undifferentiated and differentiated neuronal cells (30), possibly by hormetic growth stimulation (31). Whether phenylpropanoids can induce hormetic growth stimulation to alleviate UVC-induced skin damage needs to be investigated. Such an experiment is possible using the present irradiation system with culture medium, but not that with PBS, which cannot maintain cell survival for a long time.
The above experiments were performed using a short UVC exposure time, followed by long incubation in fresh culture medium without test compounds. It is important to monitor cytotoxicity that may appear during long incubation of cells. It was unexpected that lignified materials (Group II) such as lignosulfonate B and alkali-lignin showed very weak anti-UVC activity. On the other hand, alkaline extract such as SE showed slightly higher anti-UVC activity. This is possibly due to the presence of p-coumaric acid (degradation product of lignin) identified by metabolomics analysis (20).
Under the experimental conditions used in our study, dectin 1 protein was not detected in COLO679 cells (Figure 6). It has been reported that high dectin-1 expression is associated with shorter survival of patients with cancer (32), while low dectin-1 expression is associated with higher risk of susceptibility to fungal pathogens, graft dysfunction or death, as well increased mortality (33). These data suggest an inverse relationship between dectin-1 expression and cell survival. Further studies with more sets of malignant and non-malignant cells are necessary to confirm this hypothesis.
In conclusion, the present study demonstrated a higher anti-UVC activity of phenylpropanoids, degradation products of lignin, compared to lignin. Further studies are necessary to elucidate the mechanisms of their prominent anti-UVC activity.
Acknowledgements
This work was supported by Miyata Research Fund B.
Footnotes
Authors’ Contributions
HS, SA, SU MI, AS, and MK performed the experiments of the present study. HS wrote the article. ST, YK, MH and TO reviewed the article. HS provided interpretation of experimental results and edited the article. All Authors read and approved the final version of the article.
Conflicts of Interest
YK is employed by Nippon Paper Industries Co., Ltd., Japan. MH is employed by Daiwa Biological Research Institute Co., Ltd., Japan. The corresponding Author (HS) received the supply of purified samples of lignosulfonate B and SE from these companies. The Authors confirm that such support did not influence the outcome of experimental procedures. The other Authors declare no conflicts of interest.
- Received August 21, 2022.
- Revision received September 4, 2022.
- Accepted September 27, 2022.
- Copyright © 2022, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved
This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY-NC-ND) 4.0 international license (https://creativecommons.org/licenses/by-nc-nd/4.0).
