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Investigation of Antioxidant and In Vitro Wound Healing Activity of Fulvic Acid

This study aimed to investigate the antioxidant and in vitro wound healing activities of fulvic acid (FA) purified from Husamlar leonardite in Mugla/Turkey. Purified FA was analyzed with FT/IR spectrophotometer and flame photometer for determining functional groups and cation impurities, respectively. 1, 5, 10, 20, 30, 50, 100 μg mL-1 concentrations of FA were tested for antioxidant activity (by using DPPH radical scavenging and H2O2 scavenging assays). Also, effect of FA on BJ human foreskin fibroblast and HaCaT spontaneously immortalized non-tumorigenic human keratinocyte cells proliferation was tested by in vitro MTT and WST-8 assays and on cell migration by wound healing assay (scratch assay). The results of the study show that the FA has a low DPPH radical scavenging activity, but it exhibited high H2O2 scavenging activity at low concentrations. The effect of FA on the proliferation of BJ and HaCaT cells varied according to the cell type, FA concentration and treatment time. However, it accelerated wound healing by increasing cell migration, especially in HaCaT cells. 1 and 10 μg mL-1 FA had a significant wound healing effect on BJ cells, and all concentrations of FA had a significant wound healing effect on HaCaT cells at the end of the 24 h treatments.


INTRODUCTION
Skin is the largest organ in the body comprising approximately 15% of body weight and consists of epidermis and dermis. Furthermore, subcutaneous layer is present under the dermis. Epidermis is the outermost layer of the skin and it maintains a vital barrier against external trauma. The major cellular content of the epidermis is keratinocytes (approx. 95% of the epidermis) and fibroblasts are major cellular component of the dermis (Zouboulis, 2000;Braun-Falco et al., 2000). When the outer covering of the skin, the epidermis, is injured a wound healing response is initiated. Wound healing is a complex process that occurs in different stages and involves many different cell types (Martin, 1997). Wound repair requires the proliferation and migration of fibroblasts and keratinocytes. These cells re-establish the normal cellular and extracellular matrix composition of skin, and the growth of vascular endothelial cells (VECs) to form new blood vessels that supply nutrients to the skin cells (Barrientos et al., 2008). The ability of these three cell types (fibroblasts, keratinocytes and VECs) to repair skin integrity and function is jeopardized in nonhealing wounds (Schafer and Warner, 2008).
Humic substances are formed from the decomposition of plants and occur naturally in water, peat, soil and brown coal. Humic substances have a complex structure and can be fractioned into humines, humic acids and fulvic acids (MacCarthy, 2001). Fulvic acid (FA), a class of compounds of humic substances, is a mixture of polyphenolic compounds formed through the degradation of organic substances such as plants, microbes and animals by chemical and biological processes (Motojima et al., 2011). FA has been reported recently to have nutraceutical properties and physiological action on the human body (Winkler and Ghosh, 2018). It is one of the naturally occurring phytochemicals with neuroprotective effect (Cornejo et al., 2011;Guzmán-Martinez et al., 2013), antimicrobial and anti-inflammatory properties (Van Rensburg et al., 2001;Sherry et al., 2013).
Although with the presence of information about neuro-protective, antioxidant, antimicrobial and anti-inflammatory properties of various FA products and derivatives, no data have been reported previously on the in vitro wound healing activities of FA. Therefore, the aim of our study was to investigate the antioxidant and in vitro wound healing activities of FA purified from Hüsamlar leonardite in Muğla/Turkey on BJ human foreskin fibroblast cells and HaCaT spontaneously immortalized non-tumorigenic human keratinocyte cells.

Fulvic acid (FA) purification from Leonardite and FT/IR analysis
In this research, a method of purification process of fulvic acid was aimed. Thus, two different processes yielding calcium fulvate and ethyl fulvic ester were developed. FA is purified by method of extraction, coagulation, vacuum distillation, esterification, solid-liquid seperation and ion exchange respectively (Smith and March, 2001). In experimental studies, produced amounts of calcium fulvate and ethyl fulvic ester from 100 g of Muğla Milas Hüsamlar leonardite were 832 mg and 764 mg, respectively. After purification of these products, remaining amount of pure fulvic acid produced from ethyl fulvic ester and calcium fulvate were 800 mg and 739 mg, respectively. Thus, products were analyzed with FT/IR spectrophotometer in order to determine functional groups, and flame photometer was used for determining cation impurities ( Fig. 1 and Fig. 2). After purification studies at laboratory scale, leonardite operating plants with a capacity of 1 tonne per hour were designed for each method. 14 kg/tonne raw material calcium fulvate was produced using Özlem Sultan ASLANTÜRK et al.

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Investigation of antioxidant and in vitro wound healing activity of Fulvic acid Muğla Milas Hüsamlar leonardite (Sönmez 2011). Purified FA has carboxylic acid functional groups. Carboxyl acid groups are bonded with calcium and magnesium in coagulation phase of production process, and these ions are exchanged with sodium and hydrogen respectively. Carboxyl acid salts of calcium, magnesium, sodium and potassium are changed to hydrogen with ion exchange stages of production. After these processes, the end product is two seperate FA components: Esterificiation product FA and Calcium salt precipitation product FA. Fulvic acid purchased from International Humic Substances Society (IHHS) was used as standard agent. The free radical scavenging activity of FA was tested for its ability to bleach the stable 1,1diphenyl-2-picryl-hydrazyl radical (DPPH) (Brand-Williams et al.,1995). One milliliter of 0.1 mM DPPH methanol solution was added to 3 ml of 1, 5, 10, 20, 30, 50, 100 µg mL -1 concentrations of FA. The mixture was shaken vigorously and left at room temperature. After 30 min, the absorbance of mixture was measured at λ=517. Tests were carried out in triplicate. Ascorbic acid (10µg mL -1 ) and Rutin (10µg mL -1 ), a citrus flavonoid glycoside, were used as standard.

H 2 O 2 scavenging assay
Hydrogen peroxide scavenging activity of FA was determined by the method described by Ruch et al. (1989). A solution of H 2 O 2 (40mM) was prepared in phosphate buffer (pH 7.4). Reaction mixtures contained 40mM of H 2 O 2 and different FA concentrations (1, 5, 10, 20, 30, 50, 100 µg mL -1) , and absorbance values were measured after 10 min using wavelength of 230 nm. Ascorbic acid was used as the standard.

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Investigation of antioxidant and in vitro wound healing activity of Fulvic acid treated with 1, 5, 10 and 20µg mL -1 FA for 24h and 48h. Negative control cells received only culture medium. Crystalin (10%) was used as standard wound healing agent for BJ and HaCaT cells. 5µg mL -1 citric acid was also used as a solvent control (solvent of FA). At the end of the treatment times, cellular viabilities were determined by MTT assay (Mossman 1983) and WST-8 assay (Tominaga et al. 1999). The absorbance was read at 570 nm for MTT assay and at 450 nm for WST-8 assay by using a microplate reader (Thermo Labsystems Multiscan Spectrum).

Wound healing assay (Scratch assay)
The wound healing assay was used in this study, because it is a surrogate method for evaluation in vivo tissue healing assays and also allows the researcher to study cell migration and cell interactions (Hoang et al., 2000;Liang et al., 2007). BJ and HaCaT cells were plated in a 24well plate (10x10 3 cells per well) in Eagle's minimal essential medium containing 10% fetal bovine serum. After 24 h of cell culture at 37 ºC and 5% CO 2 , the 100% cell confluence was observed before the scratch assay was performed. A sterile yellow (100µL) pipette tip was used to make a straight scratch on the monolayer of cells, stimulating a wound. Wounded monolayers were washed twice with phosphate buffered saline (PBS) to remove cell debris. Then, cells were treated with medium containing FA at different concentrations (1, 5, 10, 20µg mL -1 ), which were determined according to proliferation assay results. Negative control cells received only culture medium. Furthermore, wound healing agent Crystalin (10%) was used as standard wound healing agent for BJ and HaCaT cells. 5µg mL -1 citric acid was used also as a solvent control (solvent of FA). Wound healing by migrating cells after FA treatment was observed at 0, 12, and 24 th hours by inverted light microscope (Olympus CK40) equipped with a camera. Photographs were analyzed by using Image J software (NIH, USA). The % relative migration of BJ and HaCaT cells treated with FA and controls were calculated according to the following equation: (1)

Statistical analysis
All the experiments were carried out triplicate, and obtained data were analyzed by One Way ANOVA (SPSS 20.00 software package program). The statistically significant difference was considered to be p<0.05. The normality of variables was evaluated using the Kolmogorov -Smirnov Z test. The statistical differences between the control and treatment groups were carried out using the non-parametric Mann Whitney Test (for independent samples). The correlations between different variables were determined using the Spearman Rank Correlation Test.

DPPH radical scavenging activity
The data on the DPPH radical scavenging activity of the fulvic acid (FA) are given in Table 1. Tested concentrations of FA exhibited very low DPPH radical scavenging activity. The DPPH radical scavenging activity of the 100µg mL -1 FA (the highest concentration in experiment) was determined as 12.37%. DPPH radical scavenging activity of citric acid, and rutin and ascorbic acid are 24.22%, 91.75% and 90.95%, respectively. FA, a soluble class of humic substances at each pH value, is a mixture of polyphenolic acid compounds that occur as a result of chemical and biological degradation of organic materials such as dead plants, microbes and animals (Motojima et al., 2011;Guzman-Martinez et al., 2013). There are studies to investigating antioxidant activity of FA obtained from different natural sources e.g. FA isolated from dissolved sludge, FA purified from coal mines in China, and FA isolated from turf (Ueda et al., 2004;Tachibana et al., 2004;Motojima et al., 2011;Gao et al., 2018). In our study, although FA showed high H 2 O 2 scavenging activity in comparison with control, it exhibited low DPPH radical scavenging activity.
DPPH can only be soluble in organic solvent and interference of absorbance from the sample compounds could be a problem for quantitative analysis (Arnao, 2000). In addition, the spectrophotometric measurements can be affected by compounds, such as carotenoids, that absorb at the wavelength of determination as well as by the turbidity of the sample (Apak et al., 2013). FA solution has a dark yellow to brown color. Although the color is very light at low concentrations, the color is getting darker at higher concentrations. As the color of the FA solution could be interfered, the measured absorbance values in DPPH and H 2 O 2 assays may be high. Interference of color of FA may have led to a low rate of antioxidant activity.

Effect of fulvic acid on the proliferation and viability of BJ and HaCaT cells
Effect of FA on the proliferation and viability of BJ cells are present in Table 2. MTT test results showed that 20µg mL -1 FA treatment decreased cell viability and proliferation significantly in comparison with control (p <0.05). However, other FA concentrations have not significantly effect on BJ cell viability and proliferation. Citric acid and Crystalin (10%) also have not an important effect on cell viability and proliferation.
According to WST-8 test results, it was observed that the 1 and 5µg mL -1 FA increased the proliferation of BJ cells at 24 h, but reduced the proliferation after treatment for 48 h. The decrease in cell proliferation after 48 h treatment of 5 and 10μg mL -1 FA was statistically Özlem Sultan ASLANTÜRK et al.

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Investigation of antioxidant and in vitro wound healing activity of Fulvic acid significant (p <0.05) in comparison with control. 20µg mL -1 FA treatment for 24 and 48 h decreased cell viability and proliferation significantly (p <0.05). Citric acid and Crystalin (10%) decreased cell viability and proliferation insignificantly. Results of the MTT and WST-8 assays exhibited that 20µg mL -1 FA significantly reduced viability and proliferation of BJ cells. Results about effect of FA on proliferation and viability of HaCaT cells are presented in Table 3. MTT test results showed that FA treatment decreased cell viability and proliferation concentration dependently. After 24h treatment, decrease of cell viability and proliferation was found statistically significant at all FA concentrations. 20µg mL -1 FA decreased cell viability and proliferation statistically significant after 24 and 48 h treatment (p <0.05).
According to WST-8 test results, it was observed that none of the FA concentrations affected HaCaT cell viability and proliferation significantly, in comparison with control groups. Citric acid and Crystalin decreased cell viability and proliferation after 24 h, but they increased cell viability and proliferation after 48 h treatment.

Wound healing effect of fulvic acid in BJ and HaCaT cells
Data for wound healing efficacy of FA on BJ cells are presented in Table 4 and Figure 3. When obtained data is compared with the control group, it is observed that crystalin has a considerably high wound healing effect at 24 th h and this effect is considerably higher than that the negative control group. However, when the effect of crystalin is compared to the citric acid, it appears that the wound healing effect of citric acid is higher than that of crystalin. 1µg mL -1 FA has significantly higher wound healing effect than the negative control at 12 th and 24 th h but, this effect is lower than that of crystalin and citric acid.  5 and 10µg mL -1 FA remained far behind the control for the first 12 h and did not show any significant wound healing effect. However, at 24 h, it surprisingly showed a significant wound-healing effect compared to the negative control. 20µg mL -1 FA did not show any significant wound healing effect on BJ cells. The citric acid showed considerable wound healing effect from the 12 th h and reached a high value (62.55%) at 24 th hour compared with negative control group, and FA and crystalin (p <0.05).  Table 5 and Figure  4. According to the findings, all concentrations of FA exhibited wound healing effects at a significant level on HaCaT cells relative to the negative control group at the 12 th h. The wound healing effects of the other FA concentrations except 10µg mL -1 were found to be higher than that of citric acid and crystalin. At 24 th h, all concentrations of FA showed significant wound healing effects in HaCaT cells in comparison with the negative control group, citric acid and crystalin (p<0.05). The most effective concentration of FA was 1µg mL -1 FA on HaCaT cells at the end of the 24-hour treatment period.  Wound healing is a highly complex biochemical event that develops in the injured organism. Disruption or prolongation of any stage of wound healing causes the wound to become chronic, which may not be delayed or improved in wound healing (Guo and Di Pietro, 2006). In our study, FA increased wound healing in both BJ and HaCaT cells related to the duration of administration. The healing process requires extra oxygen, and this demand appears in the first minute after wounding due to phagocytosis, the main event in wound healing process, which is very oxygen-consumptive (Jurcsik, 1994). Humic acid (HA) can increase the production of active oxygen during the wound healing process and in anti-tumor process. Reports by Jurcsik have shown that when HA was incubated with Hep-2 cancer cell line for 24 hours, the proliferation of the HEp-2 cancer cell line was found to be decreased by 65% as compared to the control cell line (68). HA can inhibit the tumor cell multiplication by Özlem Sultan ASLANTÜRK et al.

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Investigation of antioxidant and in vitro wound healing activity of Fulvic acid intercalating with DNA, blocks the DNA opening and destroys the DNA by producing the reactive oxygen derivatives (Jurcsik, 1994). Fulvic acid may also increase wound healing with a mechanism similar to humic acid.
In the elderly people and diabetic patients, it is often the case that the above-mentioned stages of healing of wounds become late. Oxidative stress is also one of the factors that contribute to impaired wound healing in nonhealing wounds (Schafer and Warner, 2008). Endogenous antioxidant's levels such as glutathione and vitamin E are diminished under conditions that impair wound healing including aging and diabetes (Rasik and Shukla, 2000;Ullah et al., 2016). Therefore, the wound healing potential of exogenous antioxidants has received an increasing interest.
Fibroblast proliferation and migration are important factor in wound healing. This event plays an important role in the healing process by initiating the proliferative phase of repair (Fronza et al., 2009;Ebeling et al., 2014;Ascione et al., 2016). The results of proliferation tests (MTT and WST-8) of BJ and HaCaT cells were found different from the wound healing assay results. Cell division (proliferation) is an irreversible process, and it is started by a single and rapid event, while migration is a reversible process (De Donatis and Cirri, 2009). This may be the reason for the difference between the results of cell proliferation and wound healing.

CONCLUSION
The results from our study show that fulvic acid purified from Hüsamlar Leonarditis exhibit a low DPPH radical scavenging activity, but exhibited high H 2 O 2 scavenging activity at low concentrations. Fulvic acid accelerated wound healing especially in HaCaT cells by increasing cell migration, in comparison with negative control, citric acid, and wound healing agent crystalin. Therefore, fulvic acid may be a promising agent for wound healing efficacy, especially at low concentrations. However, in order to reach a more accurate judgment in this regard, more detailed studies should be carried out using different test systems, including in vivo tests.