Cytoprotective activity of carrot and tomato callus extracts and the expression of cytokines in UV-B irradiated fibroblast cells

Studies have suggested that both carrot (Daucus carota L.) and tomato (Solanum lycopersicum L.) callus extracts contain antioxidant compounds that might have the potential to protect cells from free radicals such as H2O2 that contribute to cell damage. The other sources of free radical exposure in human cells, such as UV‐B, should also be examined. UV‐B exposure can trigger increased expression of inflammatory cytokines such as cyclooxygenase‐2 (COX‐2) and tumor necrosis factor‐α (TNF‐α) and the anti‐inflammatory cytokine interleukin‐10 (IL‐10), which causes photoaging. This study was conducted to investigate the cytoprotective activity of carrot and tomato callus aqueous extracts by observing cell viability using the MTT assay. Immunocytochemistry methods were used to examine the effects of carrot and tomato callus aqueous extracts on the expression of COX‐2, TNF‐α, and IL‐10 in human dermal fibroblast adult (HDFa) cells exposed to UV‐B light. Carrot and tomato callus aqueous extracts were obtained by the maceration method using aqua bidistilled solvent. Results showed that both carrot and tomato callus aqueous extracts at 0.5 mg/mL exhibited the highest cytoprotective effect in HDFa cells compared to that at other concentrations. Both carrot and tomato callus aqueous extracts could also decrease the expression of COX‐2 and TNF‐α, whereas carrot callus aqueous extract increased the expression of the anti‐inflammatory cytokine IL‐10 in HDFa cells.


Introduction
Aging is a physiological process that naturally occurs in the human body. It can also be accelerated by environ mental factors such as UVB exposure. Chronic expo sure of UVB light to human skin can induce the pro duction of reactive oxygen species (ROS), which con tributes to the phenomenon of skin aging. ROS in the skin also has an ability to induce inflammatory reactions through the expression of inflammatory cytokines such as cyclooxygenase2 (COX2), tumor necrosis factorα (TNFα), interleukin6 (IL6), and interleukin10 (IL10) (GrandjeanLaquerriere et al. 2003; Kanagalakshmi et al. 2014. The expression of these proinflammatory cytokines in cells can stimulate dermal matrix degradation and accel erate skin aging, whereas the antiinflammatory cytokine IL10 can prevent cells from further inflammatory reac tions (Liechty et al. 2000; Kammeyer andLuiten 2015). Schmid et al. (2008) investigated a plant stem cell ex tract derived from an apple tree type, Uttwiler Spätlauber, that had the ability to induce regeneration of human skin and hair. Plant stem cells can also be defined as callus, which is a mass of undifferentiated plant cells formed as a response to wounding. Previous studies have reported that both carrot and tomato callus extracts had higher antiox idant activities than tuber extracts (Hana 2016; Khristina 2017 and were also suggested to contain antioxidants such as flavonoids, phenolic compounds, and terpenoids. Fur thermore, carrot callus extract contains proteins such as albumin and glycoprotein, and tomato callus extract con tains unknown proteins with molecular weights ranging from approximately 19 to 108 kDa (Hana 2016; Sekar 2016; Prastowo 2017. Previous research has also demon strated the cytoprotective activity of carrot (Daucus carota L.) and tomato (Solanum lycopersicum L.) callus extracts in human dermal fibroblast adult (HDFa) cells against H 2 O 2 exposure through the restoration of the G 0 /G 1 cell cycle phase (Prastiandari 2018; Utama 2018. Further more, tomato callus aqueous extract was found to exhibit a higher rate of cell death inhibition induced by H 2 O 2 rad ical exposure than the ethanolic extract (Dewi 2018).
In this in vitro study, we reinvestigated the cytopro tective activity of both carrot and tomato callus aqueous extracts in HDFa cells irradiated by UVB exposure in pre venting cell damage by observing viable cells and the ex pression of COX2, TNFα, and IL10.

Preparation of Carrot and Tomato Callus Aqueous Extracts
Tomato ("Permata" variant) and carrot ("New Nantes" variant) calli were collected from the Biotechnology Labo ratory, Faculty of Biology, Universitas Gadjah Mada. The calli of both plants were harvested at the age 30 days un til 50 days after culturing the carrot and tomato explants in Murashige-Skoog (MS) medium supplemented with α naphthalene acetic acid (NAA) and 6benzylaminopurine (BAP) at ratios of 1:5 and 10:1, respectively. The calli of both plants were prepared by weighing and macerating in aqua bidistilled water (1 g wet tissue per 5 mL aqua bidistilled water) at 4°C for 3 h. The macer ates were then centrifuged at 12000 rpm for 12 min. The remaining solution (supernatant) was later freezedried to remove the solvent.

Analysis of Cytoprotective Effect by MTT Assay (Cellular Viability Assay)
The HDFa cultures were seeded at a density of 2×10 4 cells/well in a 96well plate and then incubated overnight in an incubator at 5% CO 2 and 37°C. Next, the cells were treated with either the carrot or tomato callus extract at three different concentrations and incubated for an addi tional 4 h. After irradiation, the medium was replaced with PBS solution and irradiated with a UVB lamp (FS40T12 UVBBP Light Sources) at 60 mJ/cm 2 for 198 s. Immedi ately after irradiation, the PBS solution was replaced with the medium for cell culture, and the cells were incubated for 24 h. After this procedure, the cell viability was ana lyzed using the MTT assay protocol (ATCC 2011), and the absorbance was measured at 550 nm. Cells incubated only with control medium were considered to be 100% viable.

Analysis of COX-2, TNF-α, and IL-10 Expression in HDFa Cells Using Immunocytochemistry Method
The HDFa cultures were seeded at a density of 5×10 4 cells/well in a 24well plate. For immunocytochemistry analysis, cover slips were placed on each well, and the cells were transferred onto the cover slips and then incu bated for 3-30 min in an incubator at 5% CO 2 and 37°C to enable adhesion of cells. Cells were treated with either the carrot or tomato callus extract at the concentration that provided the highest viability of cells in a previous exper iment conducted using the MTT assay and then incubated for an additional 4 h. Next, the cells were irradiated with UVB light at 60 mJ/cm 2 and incubated for 24 h. Fixation was done by adding methanol for 5 min. Next, 3% H 2 O 2 (blocking solution) and blocking serum were added to each well subsequently and incu bated for 5-10 min. For immunolabeling, the cells were incubated with the primary monoclonal antibody specific for COX2, TNFα, and IL10 for 30-45 min, and after two times washing with PBS, a biotinylated secondary antibody (UltraTek antipolyalent) and a streptavidinperoxidase complex reagent (UltraTek HRP) were applied according to the manufacturer's protocol. After several times of washings at each step with PBS (pH 7.4), DAB solution was added and incubated for 1-5 min, followed by counterstaining with Mayer hematoxylin. Then, cover slips were placed on each well and dissolved at increasing concentrations of alcohol for 4 min, and finally, the cells were mounted onto slides using the mounting media. The cell preparations were observed under a microscope, and the images were analyzed for cytokine expression using the ImageJ software.

Preparation of Carrot and Tomato Callus Aqueous Extracts
The process of carrot and tomato callus extraction resulted in percentage yields of 1.16% and 1.18%, respectively. The physical characteristics of the callus showed varia tions in color from whiteyellowish, whitegreenish, and slightly brownish, with a friable texture and were compact. The extracts of both carrot and tomato calli obtained from the maceration procedure were processed for further test ing in this study.

Analysis of Cytoprotective Effect of Carrot and Tomato Callus Aqueous Extracts by MTT Assay
The cytoprotective test was used to evaluate the ability of the callus extract to protect HDFa cells from UVB ex posure. The cytoprotective effect was determined by the MTT assay based on the formation of formazan crystals measured at an absorbance of 550 nm. The cytoprotective effect of the callus extract was evaluated at three different concentrations on the basis of a previous study, i.e., 0.05, 0.25, and 0.5 mg/mL for the carrot callus extract and 0.15, 0.5, and 1.0 mg/mL for the tomato callus extract (Rumiyati et al. 2019).
The results showed that UVirradiated cells exhibited an inhibitory effect by resulting in 82.36% ± 1.30% of cell viability. Pretreatment with the carrot callus extract at 0.05, 0.25, and 0.5 mg/mL resulted in cell viabilities of 93.84% ± 0.87%, 90.97% ± 0.66%, and 97.24% ± 4.05%, respectively ( Figure 1). Pretreatment with the tomato cal lus extract at 0.15, 0.5, and 1.0 mg/mL resulted in cell viabilities of 92.56% ± 0.79%, 103.77% ± 1.77%, and 97.45% ± 1.44%, respectively (Figure 2). These results indicated that both carrot and tomato callus extracts had FIGURE 2 HDFa cellular viability analysis with tomato callus extract in aqueous solution. The results showed that UV-B exposure significantly decreased the cell viability compared with untreated cell ( # p<0.05) and tomato callus extract with varying concentrations significantly increased the cell viability if compared with 60 mJ/cm 2 UV-B irradiated cells without pre-treatment extract (*p<0.05). Data are shown as a percentage of cell viability ± standard error based on one time experiment using three wells. the ability to increase the cell viability compared with the UVirradiated cells without pretreatment with the extract. Both carrot and tomato callus extracts at the concentration of 0.5 mg/mL resulted in the highest percentage of viable cells, and hence, it was used as a reference concentration for the immunocytochemistry testing analysis. A previ ous study had also demonstrated that carrot callus extract at 0.5 mg/mL resulted in the highest percentage of viable cells induced by H 2 O 2 radicals (Prastiandari 2018). Fur thermore, a concentration higher than 0.5 mg/mL, i.e., 1.0 mg/mL, had a cytotoxic effect on fibroblast cells induced by H 2 O 2 (Khristina 2017).
Based on these findings, it can be suggested that both carrot and tomato callus extracts could contain compo nents that either can act as antioxidants or have regenera tive potential. It has been reported that the callus contains a protein component similar to that in animal cells, which is known as a retinoblastomarelated protein that participates in cell proliferation and division (Desvoyes et al. 2013). Furthermore, earlier research has demonstrated that the carrot callus aqueous extract contains glycoprotein com pounds with molecular weights of 52.49 and 61.52 kDa that have been reported to possess antioxidant and anti aging properties (Lee et al. 2015; Sekar 2016. Previous

COX-2 expression ± SE (%) TNF-α expression± SE (%) IL-10 expression± SE (%)
Untreated cell 0 ± 0.00 a 0.04 ± 0.04 a 0.14 ± 0.10 a 60 mJ/cm 2 UV-B-irradiated cell 20. The results showed that UV-B exposure significantly upregulated the expression of COX-2, TNF-α, and IL-10 in HDFa cells compared with the untreated cells ( b,a p < 0.05). Carrot and tomato callus extracts at 0.5 mg/mL significantly decreased the expression of COX-2 and TNF-α and also increased the expression of IL-10 in HDFa cells compared with 60 mJ/cm 2 UV-B-irradiated cells (  studies have also identified the antioxidant compounds in both extracts and suggested the presence of flavonoids, ter penoids, and phenolic compounds in the ethanolic extract (Sekar 2016; Prastowo 2017; Trehan et al. 2017, wherein the flavonoids and phenolic compounds were found to contribute to the antioxidant activity of the tomato callus extract that is known to be soluble in an aqueous solvent.

Analysis of COX-2, TNF-α, and IL-10 Expression in HDFa Cells by Immunocytochemistry Method
The UV exposure is a potent inflammatory agent that has an ability to stimulate the production of inflammatory cy tokines such as COX2, TNFα, IL16, and IL10 (Kana galakshmi et al. 2014; Kammeyer andLuiten 2015). In this study, using HDFa cells, we evaluated the effect of both carrot and tomato callus extracts in modulating the expression of COX2, IL10, and TNFα after irradiation by UVB light at 60 mJ/cm 2 . It was observed that the cells showed positive expression of COX2, TNFα and IL10 as indicated by a brown color in the cellular cyto plasm, as depicted respectively in Figures 3-5. Further more, as shown in Table 1, both carrot and tomato callus extracts had the ability to decrease the expression of COX 2 and TNFα, as analyzed by the semiquantitative method using the ImageJ software. UVB light exposure signif icantly upregulated the expression of COX2 and TNF α in the HDFa cells to 20.61% ± 2.90% and 97.71% ± 1.34%, respectively, which is because both COX2 and TNFα are proinflammatory cytokines that are stimulated in cells by ROSinduced UVB exposure due to the activa tion of mitogenactivated protein kinase (MAPK) signal ing. Studies have reported that activation of the MAPK signaling pathway can increase the phosphorylation of P38, JNK, and ERK and lead to the production of NFκβ transcription factor, thereby regulating the transcription of proinflammatory cytokines (Surowiak et al. 2014; Subedi et al. 2018. Proinflammatory cytokines can promote the breakdown of matrix components in fibroblast cells by stimulating the infiltration of immune cells, such as neu trophils, that can damage elastin and collagen in the fi broblast skin layer due to the accumulation of matrix met alloproteinases (MMPs) 1, 2, 3, 8, and 9 (Kammeyer and Luiten 2015). Pretreatment with the carrot callus ex tract significantly decreased the expression of COX2 and TNFα in HDFa cells to up to 17.73% and 27.63%, re spectively. Similarly, pretreatment with the tomato callus extract also significantly decreased the expression of both COX2 and TNFα to up to 17.76% and 61.51%, respec tively, compared to that in UVBirradiated cells without pretreatment with the extract.
Regarding IL10 expression in HDFa cells, we found a significant increase in its expression in UVBirradiated cells to 6.03% ± 0.26%. IL10 is an antiinflammatory cytokine expressed in both immune and nonimmune cells and has a role in immunosuppression reaction. The in crease in IL10 expression after being induced by UVB in HDFa cells can be possibly regulated by STAT3 acti vation by UVB exposure, which has an important role in the IL10 cytokine activation pathway (Liechty et al. 2000; Bito et al. 2010. Pretreatment with the carrot callus ex tract was found to significantly upregulate IL10 expres sion in HDFa cells to 7.04% compared with that in UV Birradiated cells, whereas pretreatment with the tomato callus extract did not increase IL10 expression.
Results of this study indicate that both carrot and tomato callus aqueous extracts exhibited cytoprotective activities in HDFa cells exposed to UVB light by signifi cantly increasing the viability of HDFa cells and decreas ing the expression of COX2 and TNFα, whereas the car rot callus aqueous extract increased the expression of IL 10 in HDFa cells. All these findings reinforce the idea of the cytoprotective activity of carrot and tomato callus aqueous extracts that have already been evaluated in this and previous studies. Since carrot and tomato plants can be easily found anywhere in the environment, future studies can aim at developing both carrot and tomato callus ex tracts for use as antiaging agents, considering that several people are exposed to free radical sources every day. How ever, these findings still need more reinforcement studies to develop a safe and innovative cosmetic that, in the fu ture, could prevent skin damage and induce regeneration in human cells.

Conclusions
Carrot (D. carota L.) callus extract at concentrations of 0.05, 0.25, and 0.5 mg/mL and tomato (S. lycopersicum L.) callus extract at concentrations of 0.15, 0.5, and 1 mg/mL exhibited cytoprotective activity in HDFa cells against UVB exposure by increasing the viability of cells com pared to that in UVBirradiated cells without pretreatment with the extracts. Both carrot and tomato callus aqueous extracts at 0.5 mg/mL were able to decrease the expres sion of the proinflammatory cytokines COX2 and TNF α, whereas the carrot callus aqueous extract was found to enhance the expression of the antiinflammatory cytokine IL10 in HDFa cells.