Detoxification Efficiency of Micropropagated Alternanthera reineckii Briq. against Zinc Oxide Nanoparticles in Human Keratinocyte Cells

Considering the rapid developments in nanotechnology, scientific research in the field of nanotoxicology is required in order to prevent the dangers of nanotechnology on human health. For this purpose, we tested the cytotoxic effect of ZnO nanoparticle (NP), which is included in many cosmetic products, on human keratinocyte cells (HaCaT). In addition, we evaluated to potentially inhibit this cytotoxic effect with an aquatic plant, Alternanthera reineckii Briq. produced by tissue culture method. The nodal explants of A. reineckii were cultured in Murashige & Skoog basal medium (MS) including the combinations of 0.25-1.25 mg/L Thidiazuron (TDZ) and 0.25 mg/L indole-3-butyric acid (IBA). Maximum number of shoots per explant (22.50 shoots/explant) was obtained in the culture medium with 0.75 mg/L TDZ+0.25 mg/L IBA. The highest shoot length (1.77 cm) was determined in MS medium with 0.25 mg/L TDZ+0.25 mg/L IBA. Acetone and water extracts were obtained from A. reineckii through Soxhlet extraction. The cytotoxic effect of ZnO alone on HaCaT was inhibited by acetone and water extracts. The cell viability, which decreased to 26.04% with the effect of ZnO, increased up to 67.83% with the application of acetone extract. Overall, our results revealed the protective potential of this plant against nanotoxicity induced by ZnO and shed light on future studies


INTRODUCTION
In recent years, nanotechnology has appeared in many areas.Thanks to nanotechnology, analysis of nanometer-sized structures, determination of physical properties of nanometer-sized structures, and superior material production can be made (Das et al. 2019;Mohajerani et al. 2019;Jahan & Isildak, 2021).Nanosized metal and metal oxide particles are indispensable raw materials of advanced technology and their application areas are spread over many different sectors.Metal and metal oxide nanoparticles (NPs) have high catalytic, magnetic, chemical and optical characteristics.These properties vary according to the surface properties, shapes and sizes of the NPs (Kavitha et al. 2022).Zinc oxide (ZnO) is also an important NP with the aforementioned properties (Sagadevan et al. 2018).ZnO NPs can be used in sensor, surface dyes, textile products, fabrics and materials such as plastics (Uma et al. 2019;Abdullah et al. 2020;Agustina et al. 2020).Moreover, as ZnO particles become transparent when they are reduced to nano-size, they are widely preferred in personal care products, especially sun creams (Gollavilli et al. 2020).
The use of NPs in many areas has also caused their side effects.Due to the very small size of nano materials and their large surface area, they show very high chemical and biological activity.If these particles enter the body and pass for a certain time, diseases such as inflammation, wheezing and coughing may occur (Monsé et al. 2019).Ways of exposure to NPs are by inhalation, digestion, injection into the skin or body (Braakhuis et al. 2015).Especially the use of nanocosmetics has increased in recent years.However, there are risks that may occur with their increased penetration through the skin.Risk of insoluble NPs in sunscreen preparations exists (Lee et al. 2020).
It is important to include herbal extracts in the product, especially in order to reduce the risk of nanomaterials found in cosmetic products.Thus, the possibility of reducing the toxic effect of the nanomaterial will increase.Alternanthera reineckii Briq.was used as plant material in this study.A. reineckii is an aquatic plant originating from South America.This plant is in a form adapted to living in and out of water thanks to the imbalance in the current at the banks of the Amazon river (Anderson et al. 2015).A. reineckii was propagated by tissue culture techniques.Plant tissue culture is the production of plants or plant products from the whole plant or various parts of the plant under sterile conditions in an artificial nutrient medium (El-Sherif 2019; Celik et al. 2020;Ozelci & Yigit, 2022).This method is mainly based on the totipotency property of plant cells.The ability to divide while forming the complete genome of the cell is called totipotency.Apart from the totipotency feature in plants, their growth and metabolism developments are also important (Rani & Kumar 2017).Recently, tissue culture technique has been widely used in many plant-based studies such as antioxidant activity (Dilkalal et al. 2021), stress physiology (Hosseini Tafreshi et al. 2021), and secondary metabolite production (Jirakiattikul et al. 2021).We have benefited from this technique because of its advantages such as preventing the collection of plants from nature and providing rapid and multiple plant production under in vitro conditions.
To the best of our knowledge, we found that the protective effect of A. reineckii has not been tested against ZnO-induced nanotoxicity.Therefore, in the present study, we propagated A. reineckii in vitro in the desired amount and examined its protective property against cytotoxic damage induced by ZnO NPs on the human keratinocyte cells.

ZnO NPs and Their Characterization
For ZnO NPs, nano powder sample, white powder in the nanoscale range <200 nm with a high purity of 99.9+% and CAS number 1314-13-2, purchased from US research Nanomaterials Inc, Houstan, TX, USA, is used in this study.Characterization of ZnO NP is performed with X-ray diffraction (XRD) and scanning electron microscope (SEM) analysis to identify the crystal structure, the average crystallite size and morphology of NP.
In Vitro Regeneration A. reineckii plants were taken as sterile stock plants.
Murashige and Skoog (MS) basal medium with vitamins were used as nutrient media in the culture studies.The nodal explants were placed in MS medium including 30 g/L sucrose, 7 g/L agar and the combinations of 0.25-1.25 mg/L Thidiazuron (TDZ) and 0.25 mg/L indole-3-butyric acid (IBA).It was sterilized by keeping it under 1.2 atmospheres pressure at 121°C for 20 minutes.Then, the nutrient media were sterilized by keeping them under 1.2 atmospheres pressure at 121°C for 20 minutes.The culture experiment was terminated at the end of eight weeks.
In the activity studies, A. reineckii in the MS nutrient medium containing 0.75 mg/L TDZ + 0.25 mg L IBA, where the best results were obtained, were used.

Extraction
Plants (10 g) left to dry at room conditions were subjected to extraction (Soxhlet extraction).The filtered extracts were then concentrated by means of a rotary evaporator.The stock extracts obtained were dissolved with 0.5% dimethyl sulfoxide (DMSO) before the experiments.

Culture of the Cell Lines
Dulbecco's Modified Eagle Medium (DMEM) was used for culturing the human keratinocyte cell line (HaCaT).DMEM in high glucose was supplemented with 1% penicillin-streptomycin, 1% L-glutamine and 10% heat-inactivated fetal bovine serum (FBS).The cell cultures were maintained at 37°C in a humidified atmosphere with 5% CO2.

Antiproliferative Activities
Cells were seeded at 1×10 4 cells/well in 96-well flatbottomed microtiter plates.After 24 h incubation, ZnO NPs (50 mg/L) and extracts with different concentrations of A. reineckii were added to the wells alone and in combination and kept in a CO2 incubator at 37°C for 48 h.Final extract concentrations in the wells were numbered between 1-10 and showed in Table 1.Negative control (NC) cultures received 0.5% DMSO alone.MTT procedure was use for cytotoxic activity (Emsen et al. 2018

Analysis of SEM Image and XRD Spectrum of ZnO NPs
XRD patterns of resulting material in the range of 2θ = 25-75 0 was obtained (Figure 1).The well-defined sharp Bragg peaks represented extremely crystalline nature of the material with the hexagonal crystal structure (known as zincite).The particle size was changing from 37.3 nm to few hundred nm.The phase identification was made using JCPDS database and the ZnO NPs were well organized and fitted the standard of ZnO (JCPDS no: 36-1451).SEM image of ZnO NP was given in Figure 2. ZnO NPs were distributed in random as in the SEM image.2).
Shoot regeneration percentage was ranked between 66.66% and 100% (Table 2).The lowest shoot regeneration frequency (66.66%) was determined in the explants without growth regulators (control group).Mean shoot lengths ranked between 1.10-1.77cm.The longest shoot (1.77 cm) was obtained in cultures with 0.25 mg/L TDZ + 0.25 mg/L IBA, while the shortest shoot (1.10 cm) was determined in cultures with 1.25 mg/L TDZ + 0.25 mg /L IBA.

Cytotoxicity Activities
Viability rates of HaCaT cells treated with ZnO NPs and A. reineckii extracts were tested.The application that decreased the cell viability (26.04±1.09%) the most was ZnO NPs alone.AE and WE experiments alone showed a certain amount of cytotoxic effect on cells at different concentrations.While AE10 reduced the cell viability rate to 74.61±1.42%,this rate was 86.40±2.06% for WE10 application.The experimental groups with the highest cell viability among the combined AE+ZnO NPs and WE+ZnO NPs applications were AE6+ZnO (67.83±1.84%)and WE2+ZnO (65.85±2.69%),respectively (Figure 4, 5).
Heatmap and HCA analyses ranked experiments according to cell viability data and included extract and ZnO NPs experiments into different clusters.Accordingly, the experiments tested for HaCaT cells were divided into 3 clusters.ZnO NPs experiment was located under cluster 3 alone and was separated from other clusters.Applications under cluster 1 were NC and AE, WE extract trials.The combined applications of extract+ZnO NPs were placed under cluster 2 (Figure 6a).
The colour gradient appearing on the heatmap also coincided with the cluster analysis.According to heatmap analysis, ZnO NPs trial differed from other extract applications with blue color.The other extract trials, except AE9 and AE10, had red color intensity.The viability rates of cells exposed to AE9 and AE10 treatments were 78.83±1.55%and 74.61±1.42%,respectively.Combined applications, except AE8, AE9, AE10 + ZnO NPs, were in black intensity with a medium shade (Figure 6b).Pseudokirchneriella subcapitata were evaluated.The researchers exhibited that algae growth was inhibited by the increase in NP concentration, and ZnO NP caused the cell membrane to become unstable (Lee & An 2013).It is also known   When shoot lengths were examined, the highest length value was obtained in the lowest concentrations of TDZ (0.25 mg/L).The increase in the ratio of TDZ in the culture medium caused the shoots to remain short.Similarly, negative effects of TDZ on shoot lengths were previously reported by Dewir et al. (2018) and Novikova & Zaytseva (2018).It has been reported that transferring in vitro cultures to nutrient media supplemented with low concentrations of TDZ (0.01 to 1.0 μM) would be a correct solution to avoid TDZinduced adverse events such as short shoots (Novikova & Zaytseva 2018;Novikova et al. 2020).Another effective approach was to transfer the shoots in TDZ culture medium to hormone-free nutrient medium or to medium containing a plant growth regulator such as zeatin, 6-benzylaminopurine or GA3 (Sujatha et al. 2008;Dhavala & Rathore 2010).In our current study, the shoot lengths were sufficient for us, as we conducted an activity study.

CONCLUSIONS
In general, the present study demonstrated that in vitro propagated A. reineckii showed protective role against ZnO-induced nanotoxicity.Especially the combined application of acetone extract obtained from this plant and ZnO highly inhibited the ZnO-induced cytotoxic effect.This result presented the idea that the extracts of A. reineckii can be added in certain proportions to products containing ZnO used in the cosmetic field.

(
Abbreviation of the extract treatments (Özüt uygulamalarının kısaltması) Acetone/water extract at 1.95 mg/L concentration AE1/WE1 Acetone/water extract at 3.91 mg/L concentration AE2/WE2 Acetone/water extract at 7.81 mg/L concentration AE3/WE3 Acetone/water extract at 15.63 mg/L concentration AE4/WE4 Acetone/water extract at 31.25 mg/L concentration AE5/WE5 Acetone/water extract at 62.5 mg/L concentration AE6/WE6 Acetone/water extract at 125 mg/L concentration AE7/WE7 Acetone/water extract at 250 mg/L concentration AE8/WE8 Acetone/water extract at 500 mg/L concentration AE9/WE9 Acetone/water extract at 1000 mg/L concentration AE10/WE10 Statistical Analyses Differences between effects of different TDZ-IBA doses on shoot regeneration of A. reineckii from nodal explant were tested on Duncan post hoc work, an ANOVA test (p < 0.05).Hierarchical clustering and heatmap analyses were used to measure the distances between viabilities in HaCaT cells treated with ZnO NPs alone or combined with acetone and water extracts of A. reineckii.SPSS 21.0 was preferred to perform the analyses.

Figure 3 .
Figure 3.In vitro shoot regeneration of A. reineckii.Multiple shoot regeneration from nodal explants in MS medium including 0.75 mg/L TDZ + 0.25 mg L IBA after four weeks (a) and (b) eight weeks of culture Şekil 3. A. reineckii'nin in vitro sürgün rejenerasyonu.Dört hafta (a) ve (b) sekiz haftalık kültürden sonra 0,75 mg/L TDZ + 0,25 mg L IBA içeren MS ortamında nodal eksplantlardan çoklu sürgün rejenerasyonu DISCUSSION While exploring potential application areas of nanotechnology, the risks and uncertainties that NPs may pose on living things and the environment should not be ignored.The size and density of NPs play an important role in the toxic effects of NPs (Braakhuis et al. 2014).Studies on the toxic effects of nanoparticles have been investigated in many different organisms

Table 2 .
(Máthé et al. 2015)019)Z-IBA doses on shoot regeneration of A. reineckii from nodal explant Çizelge 2. Farklı TDZ-IBA dozlarının nodal eksplanttan A. reineckii'nin sürgün rejenerasyonu üzerindeki etkileriThe use of herbal products to inhibit nanotoxicity will reduce the level of side effects.Medicinal and aromatic plants used in many fields such as food, medicine and cosmetics are important in this regard(Giannenas et al. 2019).Tissue culture techniques, a current and biotechnological method, have made a significant contribution to the mass production of the medicinal and aromatic plants(Máthé et al. 2015).In order to obtain extract or active substance, the plants collected from nature can also disrupt the ecological balance.