The anti-oxidant effects of melatonin derivatives on human gingival fibroblasts

Accepted Manuscript Title: The anti-oxidant effects of melatonin derivatives on human gingival fibroblasts Authors: Chawapon Phiphatwatcharaded, Ploenthip Puthongking, Ponlatham Chaiyarit, Nutjaree Pratheepawanit Johns, Sumon Sakolchai, Pramote Mahakunakorn PII: DOI: Reference:

S0003-9969(17)30060-2 http://dx.doi.org/doi:10.1016/j.archoralbio.2017.02.022 AOB 3819

To appear in:

Archives of Oral Biology

Received date: Revised date: Accepted date:

5-12-2016 3-2-2017 28-2-2017

Please cite this article as: Phiphatwatcharaded Chawapon, Puthongking Ploenthip, Chaiyarit Ponlatham, Johns Nutjaree Pratheepawanit, Sakolchai Sumon, Mahakunakorn Pramote.The anti-oxidant effects of melatonin derivatives on human gingival fibroblasts.Archives of Oral Biology http://dx.doi.org/10.1016/j.archoralbio.2017.02.022 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

The anti-oxidant effects of melatonin derivatives on human gingival fibroblasts Chawapon Phiphatwatcharadeda,b, Ploenthip Puthongkinga,b, Ponlatham Pratheepawanit Johnsa,b, Sumon Sakolchaia, Pramote Mahakunakorna,b,*

Chaiyaritc,d,

Nutjaree

a

Faculty of Pharmaceutical Sciences, Khon Kaen University, Khon Kaen, 40002, Thailand Melatonin Research Group, Khon Kaen University, Khon Kaen 40002 c Research Group of Chronic Inflammatory Oral Diseases and Systemic Diseases Associated with Oral Health, Khon Kaen University, Khon Kaen, 40002, Thailand. d Department of Oral Diagnosis, Faculty of Dentistry, Khon Kaen University, Khon Kaen, 40002, Thailand. b

*Corresponding author at: Melatonin Research Group, Faculty of Pharmaceutical Sciences, Khon Kaen University, Khon Kaen, 40002, Thailand. Tel.: + 66 4320 2378 (office) fax: + 66 4320 2379.

Email addresses: [email protected] (Phiphatwatcharaded C),[email protected](Puthongking P), [email protected] (Chaiyarit P), [email protected] (Johns NP), [email protected] (Sakolchai S), [email protected]

(Mahakunakorn P)

2 Research highlights 

Structural modification of MLT at indole ring by adding acetyl or benzoyl groups has shown benefits in protecting oral fibroblast cells from H2O2 induced oxidative damage



The speculative mechanism to reduce oxidative damage is related with electron transfer properties that were demonstrated in TBARs, NO and cellular anti-oxidant assays.



MLT, AMLT and BMLT can be absorbed by oral fibroblast cells and the reduction of oxidative stress can be taken place inside the cells

ABSTRACT Objectives: Aim of this in vitro study was to evaluate the anti-oxidant activity of indole ring modified melatonin derivatives as compared with melatonin in primary human gingival fibroblast (HGF) cells. Methods: Anti-oxidant activity of melatonin (MLT), acetyl-melatonin (AMLT) and benzoyl-melatonin (BMLT) was evaluated by5 standard methods as follows:2, 2-diphenyl-1-picrylhydrazyl (DPPH); ferric ion reducing antioxidant power (FRAP); superoxide anion scavenging; nitric oxide (NO) scavenging; and thiobarbituric acid reactive substances (TBARs).Evaluation of cellular antioxidant activity (CAA) and protectivity against H2O2 induced cellular damage was performed via MTT assay in HGF cells. Results: According to the standard anti-oxidant assays, the antioxidant power of AMLT and BMLT were slightly less than MLT in FRAP and superoxide scavenging assays. In the NO scavenging and TBARs assays, BMLT and AMLT were more potent than MLT, whereas DPPH assays demonstrated that MLT was more potent than others. BMLT and AMLT had more potent anti-oxidant and protective activities against H2O2in HGF cells as compared with MLT. Conclusions: MLT derivatives demonstrated different anti-oxidant activities as compared with MLT, depending upon assays. These findings imply that N-indole substitution of MLT may help to improve hydrogen atom transfer to free radicals but electron transfer property is slightly decreased. Anti-oxidant and protective effects of melatonin derivatives (AMLT and BMLT) on human gingival fibroblasts imply the potential use of these molecules as alternative therapeutics for chronic inflammatory oral diseases.

Keyword: Melatonin derivatives, anti-oxidant, human gingival fibroblast cells, hydrogen peroxide, cellular antioxidant activity

3 1. Introduction Most oral diseases are associated with inflammatory response such as periodontitis, dental pulpitis, oral mucositis or recurrent aphthous stomatitis (Kim & Amar, 2006; Mehta & Kaur, 2014; Preeti, Magesh, Rajkumar, & Karthik, 2011; Prieto-Prieto & Calvo, 2004). During inflammation, the immune cells such as macrophages, neutrophils and other phagocytic cells are aggregated into the site of tissue damage and release inflammatory cytokines. Moreover, many oxidant compounds are also produced leading to oxidative stress. It is well known that inflammatory diseases are related with an increase in free radicals such as reactive oxygen species (ROS) and reactive nitrogen species (RNS) at the site of inflammation (Emelyanov et al., 2001). Self-defense mechanisms generate ROS and RNS at the site of inflamed tissues to kill pathogenic microorganisms. The inflammatory processes involve enzymes that produce free radicals such as NADPH oxidase which is capable of generating Oº-2 by enzymatically dismutating H2O2. These free radicals can disrupt the cellular functions by peroxidation. The imbalance between oxidants and antioxidants has been considered as a risk factor for chronic oral inflammatory diseases (Bagan et al., 2014; Kesarwala, Krishna, & Mitchell, 2016). It would be beneficial if we could produce the compounds containing anti-inflammatory and anti-oxidant activities for prevention or treatment of chronic inflammatory oral diseases. Melatonin (N-acetyl-5-methoxytryptamine, MLT) is a neurohormone mainly secreted by the pineal gland during the dark phase in mammals (Bonnefont-Rousselot & Collin, 2010).MLT is also found in oral compartments such as the salivary glands, saliva, and oral mucosa (Reiter et al., 2014). It was reported that decreased levels of salivary MLT were found in patients with periodontal diseases (Abdolsamadi et al., 2014; Reiter et al., 2014).MLT has anti-inflammatory and anti-oxidant activities in both in vitro and in vivo models (Cuesta et al., 2011; Guney et al., 2007; Radogna, Diederich, & Ghibelli, 2010). Production of nitric oxide (NO), prostaglandin (PGE) and inflammatory cytokines was inhibited by MLT and its metabolites in macrophage cell line (Elmegeed, Baiuomy, & Abdel-Salam, 2007; Mayo et al., 2005; Zhang, Li, Gao, & Wei, 2004). Although, melatonin has low toxicity, it also has limitation in pharmacokinetic issues such as short half-life and oral/tissue bioavailability.

In our previous studies

(Phiphatwatcharaded, Topark-Ngarm, Puthongking, & Mahakunakorn, 2014), we synthesized MLT derivatives including acetyl melatonin (AMLT) and benzoyl melatonin (BMLT) (Fig.1). These derivatives were able to reduce the production of inflammatory mediators such as NO and PGE in macrophage cells and reduced pain in rat with prolong duration of action (Rivara, Pala, Bedini, & Spadoni, 2015). Moreover, these MLT derivatives have been reported to act as a prodrug that can be cleaved to MLT by systemic enzymes (Thoại, Phạm Văn & Nam, 2013).

4 However, no studies have investigated the anti-oxidant and protective activities of these derivatives in human gingival fibroblasts (HGF). Thus, the present study compared the efficacy of MLT derivatives with MLT using five standard anti-oxidant assays and one assay for evaluating protective effect of these MLT derivatives in a primary HGF cell model.

2. Materials and Methods 2.1 Chemicals and reagents Chemicals for antioxidant assay; 2,2-Diphenyl-1-picrylhydrazyl (DPPH), fluorescein, sulfanilamide, N-(1-Naphthyl) ethylenediamine dihydrochloride (NED), phosphoric acid, phenazine methosulfate (PMS), sodium phosphate dibasic (Na2HPO4), dipotassium phosphate (K2HPO4), 2-thiobarbituric acid were purchased from Sigma-Aldrich Ltd. (USA). Nitro blue tetrazolium (NTB), nicotinamide adenine dinucleotide (NADH), 2,4,6-Tripyridyl-s-Triazine (TPTZ), iron(III) chloride hexahydrate (FeCl3•6H2O), sodium acetate trihydrate, dichloro-dihydro-fluorescein diacetate (DCFH-DA) and H2O2 were purchased from Fluka Chemika (Buchs, Switzerland) and 2,2'-azobis(2-amidinopropane) dihydrochloride (ABAP) was purchased from Calbiochem (USA). Dulbecco's Modified Eagle's Medium (DMEM), fetal bovine serum (FBS), Glutamax®, Dulbecco's PhosphateBuffered Saline (DPBS), Antibiotic-Antimycotic (100X) and Hank’s balanced salts solution (HBSS) were purchased from Gibco Inc. (USA). 3-(4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2H-tetrazolium bromide (MTT) was purchased from Fluka Chemika (Buchs, Switzerland). Melatonin derivatives were synthesized and characterizationsin a previous studyofPhiphatwatcharaded et al. (2014).

2.2 Cell culture Human gingival fibroblast (HGF) cells were kindly provided by Assist Prof. Dr. Doodsadee Homdee from Research Group of Chronic Inflammatory Oral Diseases and Systemic Diseases Associated with Oral Health, Khon Kaen University. These primary cells were cultured in DMEM with 10% FBS, 1% Glutamax® and 1% (v/v) penicillin– streptomycin at 37 °C and 5% CO2. Cells were grown to 80–90% confluence in a 10 cm cell culture dish before the experiments. Cells were counting by hemocytometer with Trypan blue staining and calculated cell number per wells before seeding into wells palte.

5 2.3 DPPH radical scavenging assay The procedure for DPPH assay was carried out according to Nicklisch and Waite (Nicklisch & Waite, 2014). Briefly, DPPH was prepared to be used in final concentration 200 μM in ethanol. The various concentrations of MLT, AMLT and BMLT (200, 600 and 1000 µM) were dissolved in DMSO and added into DPPH solution with equal volume in 96 well plates. Absorbance was recorded at 550 nm after reaction started for 30 minutes. The results are expressed as % of DPPH scavenging. DPPH solution with vehicle DMSO used as negative control, DMSO without DPPH was used as a blank for background subtraction.

2.4 Reduction of the superoxide anion radical

The reduction of superoxide anion was tested by reduction of NTB, as described by Liu and Ng (Liu & Ng, 2000). The reaction mixture contained 78 μM of NTB, 234 μM of NADH and 8 μM of PMS in phosphate buffered saline (PBS) at pH 7.4 with various concentrations of MLT, AMLT and BMLT. The reduction of NBT was measured at absorbance 550 nm after 10 min of the reaction development. The activity of the MLT, AMLT and BMLT was expressed as % of superoxide scavenging in comparison with the negative control that contained only the reaction mixture without compounds tested. The reaction mixture without NTB was used as blank for background subtraction.

2.5 The ferric ion reducing antioxidant power (FRAP) assay The FRAP assay was carried out according to the protocol previously reported by Benzie and Strain (Benzie & Strain, 1996). The FRAP reagent consists of acetate buffer (300 mM, pH 3.6), TPTZ (10 mM in HCl 40 mM) and FeCl3·6H2O (20 mM) in ratio 10:1:1. For the measurement, FRAP reagent was mixed with various concentration of MLT, AMLT and BMLT or vehicle (ethanol, negative control) as ratio 10:1. Absorbance was measured 595 nm after 10 min of the reaction development. An aqueous solution of FeSO4 (1-100 µM) was used for calibration. The results were expressed as mol of Fe2+.

2.6 Scavenging of nitric oxide free radical

6 To evaluate the nitric oxide scavenging activity, Griess's reaction was used to determine indirectly the nitric oxide production as a concentration of the nitrite end product. The method procedure was described previously by Johnson (Johnson, 1964). Sodium nitroprusside (20 mM) was freshly prepared in phosphate buffer saline (5 mM, pH 7.4) mixed with various concentrations of MLT, AMLT, BMLT (200, 600 and 1000 µM) or vehicle (ethanol, negative control) in the equal volume and allowed for incubation for 3 hours. Griess reagent (1% sulfanilamide and 0.1% NED in 2.5% phosphoric acid) was then added to each sample as ratio 1:1 and incubated another 10 minutes. Absorbance at 550 nm was recorded immediately by UV spectrometer. The results are expressed as % of inhibition of nitrite. Vehicle (ethanol without sodium nitroprusside) was used as a blank for background subtraction.

2.7 Thiobarbituric Acid Reactive Substances (TBARS) assay Mouse brain was used as source of lipid for malondialdehyde (MDA) generation in the Fenton reaction described in (Mandal, Hazra, Sarkar, Biswas, & Mandal, 2011) with some modifications. The whole brain was homogenized in 20 ml of solution containing of phosphate buffer (3.2 mM, pH 7.4) and KCl 150 mM in glass teflon homogenizer. Then, various concentrations of MLT, AMLT, BMLT or vehicle (ethanol, negative control) in a volume of 50ul were added to each 200 μl of brain homogenate in the centrifuge tubes and adjusted to 2 ml with phosphate buffer. The solution mixture were shaken and incubated at 37oC for 30 minutes except blank that was incubated on the ice bath. After incubation, 500 μl of perchloric acid was added to precipitate the proteins and centrifuged at 3000 rpm for 5 minutes. Subsequently, supernatant was collected and mixed with thiobarbituric acid (final concentration as 0.025%), incubated and shaken on water bath at 100°C for 15 minutes. Thiobarbituric acid reactive substances (TBARs) were measured via spectrofluorometer at excited wavelength 528 nm and emission wavelength 551 nm. The results are expressed as %inhibition of lipid peroxidation. Vehicle (ethanol plus phosphate buffer without brain homogenate) was used as a blank for background subtraction.

2.8 Cellular Antioxidant Activity (CAA) assay The CAA assay is a method to evaluate anti-oxidant activity at a cellular level described previously by Wolfe and Liu(Wolfe & Liu, 2007). Briefly, HGF cells were seeded for 10 x 104 cells/well on a 96-well plate. After incubating in 5% CO2and 37 °C for 24 hours, the growth medium was removed and the wells were washed by 200 µl of PBS. Each well was treated with MLT (200 µM), AMLT (200 µM), BMLT (50 µM) or vehicle (1%DMSO in DPBS,

7 negative control) in medium plus 25µM of DCFH-DA for 1 hour. After washing, PBS 100µL and ABAP600 µMin 100µL of HBSS was added into each well. Fluorescence intensity was measured at 37°C via fluorescence microplate readers at emission wavelength 538 nm and excitation wavelength at 485 nm every 1 minute for 1 hour. Quercetin (1100 µM) was used for calibration. The results were expressed as µmol equivalents of quercetin.

2.9 Protective effect against H2O2 induced fibroblast cells death Human gingival fibroblast cells (HGF) grown to 80–90% confluence in cell culture dish were counted and seeded into a 96 well-plate (10 x 104 cells/well) and incubated for 24 hours. Various concentrations of MLT, AMLT and BMLT (1% DMSO in final concentration) were added to the cells with or without 0.4 µM of H2O2. Subsequently, cells were incubated for another 24 hours. To determine cell viability, MTT assay was performed. MTT (50μg/mL) was added and cells were incubated for another 30 min. The culture medium was then removed and the cells were dissolved in DMSO. The optical densities (OD) at 540 nm were measured with a microplate reader (Bio-Rad Model 680, Tokyo, Japan). Fresh culture medium was used as a blank for background subtraction.

2.10 Statistical analysis All of experimental data will be demonstrated as the mean ± SD. Analysis of variance (ANOVA) will be used to analyze group differences between samples treated and negative control (vehicle treated only) via software SigmaStat for Windows version 3.11. A probability (P) level lower than 0.05 is indicated significance.

3. Results 3.1 Effect of MLT and its derivatives on reduction of DPPH The effect of MLT, AMLT and BMLT in various concentrations (0.2, 0.6 and 1 mM) on DPPH scavenging at 30 minutes shown in Figure 2. MLT reduced DDPH free radical in a dose-dependent manner with 6%, 13% and 16% of DPPH scavenged, respectively, whereas AMLT and BMLT did not show significant reaction with DPPH. However, MLT at highest concentration used (1 mM) still exhibit a weak efficacy to scavenging of DPPH at 30 minutes.

3.2 Effect of MLT and its derivatives on reduction of superoxide anion

8 In the PMS/NADH-NBT system, superoxide anion was generated from PMS/NADH coupling reaction reduced NBT. Scavenging of the superoxide anion by MLT was only significant at concentration 1000 μM (29% scavenging). In the same concentration range BMLT was more efficient (significant difference was seen at the concentrations of 600 and 1000 μM for 28% and 35% scavenging, respectively) while AMLT was less efficient exerting a significant effect in the highest concentration only (17% scavenging) (Fig. 3).

3.3 Effect of MLT and its derivatives on FRAP FRAP was used as the third assay to evaluating electron donating properties of MLT and derivatives. In this reaction system,MLT at 100 and 1000 µM were able to reduce the ferric tripyridyltriazine to 7µM and 18 µM ferrous form, respectively, while BMLT at 1000 µM can reduced 7 µM of ferric tripyridyltriazine. AMLT at same concentrations did not show significant activity to reduce FRAP (Fig. 4). Moreover, reduction of FRAP by MLT was much higher than by other derivatives.

3.4 Effect of MLT and its derivatives on scavenging of nitric oxide free radical The effect on nitric oxide scavenging was indirectly measured as % inhibition of nitrite (Fig.5).MLT and both derivatives were efficient in a dose-dependent manner. Among the products examined BMLT appeared to be the most potent inhibitor (in a range of 23 to 61% of inhibition) while activity of AMLT and MLT were similar at the same concentrations (in a range of 9-24% of inhibition).

3.5 Effect of MLT and its derivatives on TBARs assay In this assay (Fig.6), MLT, AMLT and BMLT at the range of concentrations of 200, 400 and 600 µM exhibited dosedependent manner to inhibit TBARs production. Among the products examined BMLT exhibited the highest activity (inhibition for 27%, 43% and 67%, respectively), and the activity of AMLT (inhibition for 19%, 34% and 48%, respectively) was also higher than MLT at same concentrations (inhibition for 25%, 29% and 33%, respectively).

3.6 Effect of melatonin and its derivatives on protection of HGF cells from H2O2

9 In this evaluation system, the result showed that BMLT at 5 µM could protect 30% of HGF cells whereas 25 µM of BMLT protected 85% of cells (Figure 7). AMLT and MLT could protect 30% of HGF cells at concentrations of 50 µM and 150 µM respectively. Moreover, at a concentration of 200 µM AMLT and MLT exhibited protective effect of 60% and 35% respectively.

3.7 Effect of melatonin its and derivatives on cellular anti-oxidant activity in HGF cells This result showed that BMLT (50 µM) could reduce oxidative stress from ABAP equivalent to 29 uM quercetin, whereas AMLT (200 uM) and MLT (200 uM) were equivalent to 25 and 10 uM, quercetin respectively (Fig. 8).

4. Discussion Our previous study demonstrated that the anti-inflammatory effects of BMLT and AMLT on production of inflammatory mediators such as nitric oxide and prostaglandin E2 in LPS-stimulated murine macrophage cell line were better than those of melatonin (Phiphatwatcharaded et al., 2014). The present study extended our previous study by investigating the anti-oxidant and protective activities of these melatonin derivatives. Regarding standard anti-oxidant assays, the DPPH, superoxide scavenging and FRAP assays were choose to evaluate electron transfer property (Rodriguez-Naranjo, Moyá, Cantos-Villar, & Garcia-Parrilla, 2012). In addition, nitric oxide scavenging and TBARs assays can act though both of electron or hydrogen atoms transfer mechanisms. Our results showed that in FRAP and DPPH models, the native MLT molecule exhibited the highest activity in comparison to the molecules with the modification at the N-indole ring, i.e. AMLT and BMLT. However, MLT still exhibited poor activity compared with ascorbic acid, vitamin E or Trolox on FRAP and superoxide scavenging assays which reacted with electron transfer mechanism (Andreadou, Tsantili-Kakoulidou, Spyropoulou, & Siatra, 2003; Fagali & Catalá, 2007; RodriguezNaranjo et al., 2012). Galano Annia (Galano, 2016) have been found the relationship between electron donor properties and antioxidant activity of new synthetic MLT derivatives in DPPH assay. Those information suggest that AMLT and BMLT have lower electron donor properties than MLT. Superoxide anion (Oº2-) is a less reactive ROS compare to OHº. However, Oº2- might eventually be more toxic due to its reactivity with nitric oxide or phenoxyl radical. Electron transfer is a rate-limiting step for the superoxide anion scavenging. Allegra et al. (Allegra et al., 2003) have reported that MLT exhibited weak scavenging of Oº2-. In our experiment, Oº2- was generated by dissolved oxygen in the PMS/NADH coupling reaction and MLT showed poor

10 scavenging potency in this system. Moreover, substitution of acetyl or benzoyl moiety group into N-indole ring of MLT did not show benefit in this assay. In the lipid peroxidation assay, brain is organ that very sensitive to oxidative damage (Pizzimenti et al., 2013). Our experiment using heat induced process of Fenton's reaction to screening effect on lipid peroxidation. This reaction can found in many tissue included to mucosa membrane. Fatty acid were reacted with peroxyl radicals which generated MDA as a end product. It have been reported that 5-methoxy group of MLT structure is important for scavenging peroxyl radicals. Modifications of N-indole ring also increase electron localized in the ring that 5-methoxy group donates electron to peroxyl radicals (Pieri, Marra, Moroni, Recchioni, & Marcheselli, 1994). Antunes et al. (Antunes et al., 1999) suggested that indole ring is not important for scavenging the peroxyl type of radicals. Accordingly, our modification showed that substitution of acetyl or benzoyl group at N-indole ring provide a benefit to protect against the lipid peroxidation. NO is involved in a variety of inflammatory conditions, neuronal function and the immune system. However, excess levels of NO lead to the pathological conditions such as cancer or chronic inflammation (Xu, Liu, Loizidou, Ahmed, & Charles, 2002). Indole ring of MLT has been shown to react with NO and NO-derived nitrogen species by nitrosation and oxidation reaction to form 1-nitrosomelatonin and 1-hydroxymelatonin (Turjanski, Sáenz, Doctorovich, Estrin, & Rosenstein, 2001). Thus, a modified MLT structure at the N-indole ring might alter NO scavenging properties. Interestingly, our experiment showed that BMLT have highest capacity to reduce NO while AMLT efficacy was close to that of MLT in sodium nitroprusside system. These results can be explain that nitrogen atom of BMLT and AMLT are amide functional group and it can donated electron to indole core structure due to more stability of indole-nitrosation binding. Additionally, using primary HGFs cells to evaluate a protective effect of MLT and its derivatives on H2O2– induced damage also have shown similar results, i.e. that BMLT and AMLT have higher efficacy than MLT. Moreover, in the cellular anti-oxidant activity assay, which is more biologically representative to pharmacokinetic process than in-vitro chemical antioxidant assays in term of metabolism and cell absorption, we have also shown the same trend of protective effects. Those results suggest that MLT and derivatives can pass through the fibroblast cell membrane to reduced oxidative stress from ROS and RNS inside the cell. HGF cells is a human primary cell which responds to induced inflammatory response by lipopolysaccharide and hydrogen peroxide. Moreover, immune function of primary cells are more truthful than immortal cells line (Kaur & Dufour, 2012). Mitochondria are one of the important organelles that produce energy from oxygen as well as ROS inside the cell. MLT and derivatives have

11 potential to protected mitochondria dysfunction from oxidative stress. However, the experiments to evaluate function of mitochondria in oxidative stress also needed in future study. Therefore, the mechanism of MLT and derivatives to act as the anti-oxidants might mainly consist of direct anti-oxidant mechanisms both inside and outside of the fibroblast cells. However, data of anti-inflammatory, anti-oxidant and protective activities of these derivatives on other oral cells such as oral epithelial cells and dental pulp cells are needed to confirm for protectivity effect of oral cavity. Modification of the MLT structure might be beneficial for its anti-oxidant activity. Many mechanism of MLT interaction with free radicals have been proposed (Allegra et al., 2003). AMLT and BMLT have been modified at Nindole which changes the property of functional group from amine to amide and alters the electron resonance of the indole ring. Due from modification, we found that AMLT and BMLT have better to transfer hydrogen atom to free radicals than MLT in non-cellular and cellular assays. Moreover, these derivatives also offer better anti-inflammatory activity than their parent compound. Nevertheless, these substitutions might be lead to steric accessibility on some type of radicals such as DPPH (Apak, Özyürek, Güçlü, & Çapanoğlu, 2016). Many study reported that MLT has less toxicity even in high doses in clinical and preclinical study; mostly adverse event is disturb sleep pattern (FerracioliOda, Qawasmi, & Bloch, 2013; Jan et al., 2000; Sainz et al., 2003; Sugden, 1983). In our experiment also found that high doses of MLT at concentration 200 µM did not affect on HGF cells viability. AMLT also showed low toxicity at the same concentration as MLT in HGF cells but BMLT was tested in lower concentration because of limited solubility, it will be precipitated and toxic to HGF cells when the concentration is higher than 50 µM. The greater lipophilicty of BMLT is however an advantage in terms of better tissue delivery and lower dose required. In our previous experiments (Phiphatwatcharaded et al., 2014), we did not noticed any acute side effect in mice after received MLT derivatives for 7 days. This data suggest that MLT derivatives might be had benefits to increase duration of action and activity but data on toxicity also needed in future study. Shim et al. (Shim et al., 2015) have been modified MLT structure at ethylamine group to 5-hydroxy-2'-isobutyl-streptochlorin. Their results showed that those derivatives also expressed much more anti-inflammatory activity than MLT. Their derivatives also demonstrated suppression of immune cells infiltration, reduced inflammatory cytokines IL-6 and TNF-α, and inhibition of NLRP3 inflammasome activation. Consequently, we need to evaluate the possible mechanism for anti-inflammatory action and effects on anti-oxidant enzymes of MLT derivatives and animal models in the future study to understanding and confirm therapeutic properties of these derivatives for treatment of chronic inflammatory oral diseases. 5. Conclusion

12 These results show that modification of MLT structure at N-indole ring can alter anti-oxidant activities. On the assays that depend on electron transfer mechanism, the activities to transfer electrons of AMLT and BMLT are slightly decreased when compared with unmodified MLT. However, the anti-oxidant activities of theses derivatives are better than MLT on radical type that need hydrogen transfer reaction to scavenging inside fibroblast cells. It has high possibility to develop those MLT derivatives as new therapeutic agents for chronic inflammatory oral diseases. In addition, future study on other oral cells or animals models would be of importance to confirm specific mechanisms and efficacy of MLT derivatives.

Ethical approval: This article has approval of the Ethics Committee for the collection of human gingival fibroblast cells from human subjects, Khon Kaen University (HE542141). Conflict of interest: The authors have declared that there is no conflict of interest. Informed consent: All participants signed consent forms.

Acknowledgments The authors would like to thank the express our appreciation to Prof. Krystyna Skwarlo for her kind suggestion regarding the preparation of this manuscript and statistical analyses. Funding/support information: Financial support from National Research Council of Thailand, Melatonin Research Group, and the Research Group of Chronic Inflammatory Oral Diseases and Systemic Diseases Associated with Oral Health, Khon Kaen University

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17

Figure legends Figure 1:

Structure of melatonin (MLT), acetyl melatonin (AMLT) and benzoyl melatonin (BMLT).

Figure 2:

Concentration-related effect of MLT, AMLT and BMLT on scavenging of DPPH free radicals. The results (means + SD, n = 6) are expressed as % of DPPH scavenging, * = P<0.05 when compared to negative control (n = 6).

Figure 3:

Concentration-related effect of MLT, AMLT and BMLT on superoxide anion scavenging in PMS/NADH-NBT system. The results (means + SD, n = 6) are expressed as % of superoxide scavenging, * = P<0.05, ** = P<0.001 when compared to negative control.

Figure 4:

Concentration-related effect of MLT, AMLT and BMLT on reducing of ferric tripyridyl triazine to ferrous form. The results (means ± SD, n = 6) are expressed as Fe2+ equivalent (µM), * = P<0.05 when compared to negative control.

Figure 5:

Concentration-related effect of MLT, AMLT and BMLT on nitric oxide free radical scavenging evaluated in the scavenging evaluated in the sodium nitroprusside. The results (means + SD, n = 6) are expressed as % inhibition of nitrite. * = P<0.05, ** = P<0.001 when compared to negative control.

Figure 6:

Concentration-related effect of MLT, AMLT and BMLT on the reduction of lipid peroxidation. The result (means + SD, n = 4) are expressed as shown as % inhibition of TBARs generation, * = P<0.05, ** = P<0.001 when compared to negative control.

Figure 7:

Concentration-related effect of MLT and its derivatives on the protection of HGF cells against H2O2 induced cytotoxicity. Cells were co-culture with various concentrations of MLT or derivatives with 400 µM of H2O2 for 24 hours. The results are expressed as %protection against H2O2 (n = 8, 3 replications).

Figure 8:

Effect of MLT and its derivatives in cellular anti-oxidant activity. Fluorescence intensity was measured kinetically for 1 hour. The results (means + SD) are expressed as molar equivalent with quercetin in cellular anti-oxidant activity (n = 8). * = P<0.05 when compared to negative control.

18

H N

H3CO N H

Melatonin

CH3

H N

H3CO

O

O

N

O

CH3

CH3

Acetyl melatonin

H N

H3CO

CH3 O

N

O

Benzoyl melatonin

19

%Scavenging of DPPH

40

30

20 *

*

10

0 0

200 600 Concentration (µM)

1000

%Scavenger of superoxide

20

60

**

40 *

* *

20

0 0

200 600 Concentration (µM)

1000

21

20

*

18

Fe2+ equivalent (uM)

16 14 12 10 *

8

*

6 4 2 0 0

10

100 Conc. (uM)

1000

%Inhibition of nitrite

22

100 90 80 70 60 50 40 30 20 10 0

** **

*

0

*

200 600 Concentration (µM)

*

*

1000

23

%inhibition of TBARs

100 80 ** 60

** *

40 *

*

*

** *

*

20 0 0

200 400 Concentration (µM)

600

24

100

% Protection agianst H2O2

90 80

MLT

70

AMLT

60

BMLT

50 40 30 20 10 0 1.25

2.5

5

12.5 25 50 Concentration (µM)

100

150

200

25

Quercetin equivalent (µM)

35 *

30

*

25 20 *

15 10 5 0 Blank

MLT 200 uM AMLT 200 uM BMLT 50 uM Compounds