Suppressive efficiency of Kojic acid from Aspergillus tamarii MM11 against HepG-2 cell line derived from human liver cancer

Purpose: To evaluate the antioxidant and cytotoxic properties of Kojic acid (KOJIC ACID) from Aspergillus tamarii MM11 against HepG-2 cell line derived from human liver cancer. Methods: The crude extract of A. tamarii MM11 was dissolved in a mixture of CH2Cl2/MeOH (85:15) and separation was done using silica gel chromatography using gradient size exclusion chromatograph. The non-polar oily fractions were subjected to gas chromatography-mass spectrometric (GC-MS) analysis. Kojic acid structure was identified by x-beam crystallography and spectroscopic methods. Total antioxidant properties of KOJIC ACID were evaluated by using 1,1-diphenyl-2picrylhydrazyl (DPPH) against ascorbic acid as a reference. The cytotoxic activity of KOJIC ACID from A. tamarii MM11 was investigated on the human cell line of liver cancer (HepG-2) using a sulforhodamine B (SRB) assay based on a cell density determination by the measurement of cellular protein content. Result: Highly bioactive Kojic acid was isolated as the main product. A. tamarii MM11 Kojic acid showed good antioxidant activity with half-maximal inhibitory concentration of IC50 at concentrations of 10.34 compared to 6.79 μg/mL for ascorbic acid. Kojic acid also showed good cytotoxic activity against HepG-2 cell line of human liver cancer with IC50 at 6.20 compared to 3.25 μg/mL of reference drug doxorubicin. Conclusion: Kojic acid produced naturally from A. tamarii MM11 shows good antioxidant and cytotoxic activity against HepG-2 cell line derived from human liver cancer. These findings suggest that Kojic acid can be therapeutically used as an antitumor drug after further in vivo studies.

Cancer is a life-threatening disease. Most of the successive anticancer medications currently used cause many undesirable side effects. For example, Doxorubicin can prompt cardiotoxicity and tumor drug resistance [3]. Methotrexate also can cause liver damage and portal hypertension and cirrhosis. While, Cisplatin administration can lead to nephrotoxicity and in some cases renal failure [4].
Therefore, new anticancer drugs with more efficiency and ability to mitigate side effects are needed.
Fungal anticancer secondary metabolites are one of the very important targets for mycologist. In this connection, 5-hydroxy-2hydroxymethyl-γ-pyrone (HMP) or Kojic acid (KOJIC ACID) is a major secondary metabolite produced by a limited range of microorganisms, including Aspergillus oryzae, A. flavus, and A. tamarii, as well as Penicillium species and certain bacteria [5].
Kojic acid possess strong antioxidant, antibacterial and antifungal activities. So, it is widely used in medical purposes and many other fields. It also used as a food flavor enhancer [6]. In agriculture, it used as anti-melanosis and insecticide activator [7]. In cosmetic, Kojic acid is well known as whitening agent, ultraviolet filter, tyrosinase inhibitor, and radio-protective agent [8]. Few studies were performed on anticancer activity of Kojic acid.
In the present investigation, most of the secondary metabolome of terrestrial A. tamarii MM11 was detected and the produced Kojic acid was isolated, purified and elucidated and its antioxidant properties were studied. Furthermore, the cytotoxic effect of Kojic acid on liver carcinoma cell lines were determined.

Fungal strain
The terrestrial fungal isolate used in this study was isolated from tubers of rotten Jerusalem artichoke (H. tuberosus) identified by molecular sequencing of fungal Inter Transcribed Spacer (ITS). The fungus strain was identified as A. tamarii MM11 with the accession no. GU295949.

Fermentation
A. tamarii MM11 was inoculated from well grown agar plates colonies in 0.1 L sterilized glass bottles each containing sterilized rice. The medium composition was: 8 g commercial rice; 10 mL distilled water. The bottles were incubated for 15 days at 28 ± 2°C. After harvesting, 50 mL of 1:1 DCM/MeOH was added to each bottle, followed by vigorous shaking for two hours. The afforded organic extract was decanted, filtered and then concentrated in vacuo till dryness.

Determination of total antioxidant activity
The antioxidant activities of fungal KOJIC ACID were detected using 1,1-diphenyl-2picrylhydrazyl (DPPH) in comparison with ascorbic acid as standard radical scavenging agent. The experiment was carried out by preparing solutions of 50 mg/mL, and then serial dilutions (5-50 mg/mL) of KOJIC ACID and the reference ascorbic acid were prepared. Then, 250 µL of each dilution was added to 1 mL DPPH solution (6 mg/50 mL). Control tube was also prepared using 1 mL of ethanol. The mixture was shaken and incubated for 30 min in the dark at room temperature. Absorbance was measured using a Genway spectrophotometer at 517 nm [9]. These steps were repeated 3 times and the radical scavenging (R) evaluated according to Eq 1.
R % = 1-(As/Ac) x 100 …………. (1) where, R is radical scavenging, As is the absorbance of the sample and Ac is the absorbance of the control.

Evaluation of cytotoxic activity
The cytotoxic activity of KOJIC ACID of A. tamarii MM11 was investigated on the human cell line of liver cancer (HepG-2) using sulforhodamine B (SRB) assay based on a cell density determination by the measurement of cellular protein content. HepG-2 monolayer was fixed on the 96-well plate with trichloroacetic acid (TCA). Then, SRB was added to each well and incubated at room temperature for 1h. SRB binds to basic amino acids in cellular proteins under mild acidic conditions. The excess dye was removed by washing repeatedly with acetic acid. The protein bound dye was dissolved by adding Tris-base solution (basic medium) to each well and shake the plate to solubilize the protein bound dye.
The amount of bound dye can be determined by measuring the absorbance at 510 nm in a microplate reader. It can then be extrapolated to measure cell proliferation [10]. HepG-2 used in this study was obtained from the American Type Culture Collection (ATCC, Minisota, U.S.A.). The tumor cell line was maintained at the National Cancer Institute, Cairo, Egypt, through serial sub-culturing. Doxorubicin was used as the reference drug.

Statistical analysis
Statistical analysis of the results was carried out using GraphPad instant, Version 3.06 (GraphPad Software Inc, San Diego, California, USA). The data are expressed as mean ± standard deviation (SD). Curves plotting were performed with Origin 6.0
According to EI MS, the molecular weight of Kojic acid was established as 142 Dalton with a corresponding molecular formula of C6H6O4. Based on the proton nuclear magnetic resonance spectroscopy ( 1 H NMR, DMSO-d6), two singlets were visible at 7.97 and 6.34, being for aromatic/olefinic attached protons, together with two broad singlets at  8.99 and 5.69 ppm being for phenolic and aliphatic hydroxyl protons, respectively, in addition to an sp 2 -attached oxymethylene protons were shown at  4.28 ppm.
On the bases of 13 C NMR/APT spectra of Kojic acid, six carbon signals, as matched with the afforded molecular formula, were deduced, being for one γ-lactone carbonyl ( 174.2), three sp 2quaternary Oxy-carbons ( 168.2 and 153.0), two sp 2 -CH carbon signals ( 138.9 and 110.0) and one sp 2 -attached Oxy-methylene (59.7). According to these data, searching in AntiBase and comparison with literature, Kojic acid structure was confirmed.
Kojic acid from A. tamarii MM11 recorded strong antioxidant activities with IC50 value reached to 10.34 µg/mL in comparison with the reference compound (ascorbic acid) which recorded IC50 of 6.79 µg/mL. These values indicated potent radical scavenging activities of KOJIC ACID (Table 3). Kojic acid also showed excellent cytotoxic activities against cancerous human liver cell line (HepG-2) with IC50 equals to 6.20 µg/mL in comparison with 3.25 µg/mL for the reference drug Doxorubicin (DOX) ( Figure 6)

DISCUSSION
The great progress in instrumental analysis devices contributed greatly in understand the secondary metabolome map of active fungi. These maps give a platform of secondary metabolites informatics which can be used in many applications. In this article, 100 organic secondary metabolites were identified using GC mass from A. tamarii MM11 extract. Generally, A. tamarii produces vast types of secondary metabolites including fumigaclavine A, aflatoxin, cyclopiazonic acid, speradine A and kojic acid. A. tamarii produces a considerable amount of Kojic acid, [13]. Many studies evaluated antimicrobial and antioxidant properties of KOJIC ACID, but only few studies investigated its anticancer activities. In the 1950s, Gerschman et al [14] demonstrated that oxygen-containing free radicals. have hazardous effects on all living cells. Reactive oxygen species (ROS) are endogenous, very active oxygen bearing atoms, which can be divided into enzymatic and nonenzymatic classes [15]. ROS have been believed to be the main cause of various diseases as cancer, sclerosis, Parkinson's, Alzheimer's, immune system ailment, stroke, and others [16].
Antioxidants are the compounds that can neutralize ROS and provide protection against cancer by lowering the peril of tumor development [17].
From the results, purified Kojic acid from A. tamarii MM11 gave relatively high DDPH radical scavenging activity. It showed potent antioxidant properties with a very close IC50 to that of the reference ascorbic acid. Kojic acid can be effectively served as nontoxic naturally occurring antioxidant, blocking the action and side effects of many routinely ROS produced during the photodynamic therapy of neoplastic diseases and others such as arteriosclerosis and diabetes [18]. As a promising result, Kojic acid showed highly cytotoxic effects on HepG-2 cells that suggesting strong antitumor effects of Kojic acid against hepatocellular carcinoma. These results were previously observed by another study indicated that Mannich indicated by a study documented that the combination therapy of Mannich base containing ciprofloxacin and Kojic acid structural units showed antitumor activity in HepG-2 [19]. Kojic acid is a potent inhibitor for cellular NF-kappaB activity in different cell types. It is documented that KOJIC ACID has this inhibitory effect in transfectant HaCaT cells, SCC-13 cells and in human keratinocytes. It was found to be more effective than other antioxidants as ascorbic acid and N-acetyl-Lcysteine which suggested that Kojic acid induced anti-melanogenic effect [20]. Previous studies showed that KOJIC ACID also inhibit cell growth of A375 melanoma cells. So, it is used now as a treatment for many types of melanoma [21,22].
There is argument about the effect of Kojic acid administration and DNA mutations. There was a study suggested the ability of Kojic acid to cause mutations in salmonella bacteria [3]. However, other in vivo mammalian studies proved KOJIC ACID as a safe drug at relatively high concentrations that is not significant acute oral toxicant in mice and rats with LD50 value greater than 1 g/kg [6,23].

CONCLUSION
The purified form of Kojic acid isolated from Aspergillus tamarii MM11 shows radical scavenging activity close to that of ascorbic acid. It also exhibits good cytotoxic activity in HepG-2 cell lines. Thus, kojic acid is a potentially safe, natural antioxidant and antitumor agent.