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-2- picrylhydrazyl (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. Keywords: Aspergillus tamarii, Secondary metabolites, Kojic acid, Anticancer, Liver cance

The Isolation Of Kojic Acid Producing Fungi And The Optimization Of Kojic Acid Production

A local strain of fungus, which was found to be a high kojic acid producer has been isolated from Morning glory flower (Bixa orellana) using glucose-peptone medium. The pure strain was obtained through several steps of monospore isolation procedures using spread plate technique and identified by International Mycological Institute as Aspergillus flavus Link.Optimization of medium composition and cultural conditions for kojic acid production by this fungus were carried out in shake flask. The development of dissolved oxygen tension (DOT) control strategies aimed at improving kojic acid production by this strain was carried out using 1.5 L stirred tank reactor with automatic pH and DOT control systems. Initial culture pH 3 was found to POLO be optimum for kojic acid production. This strain was able to grow and produce kojic acid in various carbon sources such as glucose, starch, sucrose and xylose. However, the highest production of kojic acid was obtained at 100 g/L (w/v) glucose. In addition, 5 g/L of yeast-extract was found to be the best nitrogen source for fungal growth and enhancement of kojic acid production. Addition of 4% (v/v) of methanol to glucose yeast-extract medium increased kojic acid production by two times. The optimized medium for kojic acid production for this strain was proposed and the medium consisted of 100 g/L glucose, 1.0 g/L KH2PO4,0.5 g/L MgSO4. 7H20, 5.0 g/L yeast-extract and 4% methanol. Using this optimal medium the maximum kojic acid production in batch fermentation using shake flask was 39 g/L. This fermentation gave yield and productivity of 0.04 g/g and 0.07 g/L/h, respectively and is comparable to that reported in the literature for industrial strain. The DOT control strategy for the improvement of the kojic acid production by this strain was also developed. By controlling DOT at 80% saturation during growth phase and then switched to 30% saturation during production phase, increased the production of kojic acid by about two times as compared to uncontrol fermentation in the stirred tank fermenter which only produced maximum concentration of kojic acid of 15 g/L.. The effect of the addition of yeast-extract during the fermentation was also investigated using a constant fed-batch culture. The addition of 15 g/L yeast-extract at constant flow rate of 3.2 ml/h during the production phase caused excessive mycelial growth and decreased kojic acid production

Improvement of Kojic Acid Production by a Mutant strain of Aspergillus flavus

The ability of 58 different strains of Aspergillus, Mucor, and Penicillium to form kojic acid has been studied. Four Aspergillus species and Penicillium cryzogenum local isolates with high activity of kojic acid synthesis were screened for kojic acid production on five proposed kojic acid producing synthetic medium. Aspergillus flavus (AF) was found to be the highly active one for kojic acid production. The highest level of kojic acid productivity (0.234 g/ l.h) obtained by A. flavus using fermentation medium of 100g/l glucose, and 5.0 g/l yeast extract, and incubated at 30°C and 180rpm for 9 days. The study implicated the optimization of different carbon and nitrogen source of fermentation medium. Among the carbon sources tested, glucose gave the highest kojic acid yield (50.27 g/l) followed by sucrose (48.95 g/l). The use of 5g/l yeast extract resulted in the highest kojic acid production (50.21g/l) compared with the other nitrogen sources. Improvement of Aspergillus flavus by natural selection and random mutagenesis by using UV (for 5, 10, 20, 40 and 60 min) and various doses of gamma irradiation (20, 40, 60, 80, 100, 120, 140 and 160 Gy) has been done to obtain a potential mutant which produce kojic acid higher than its parent strain. Some of wastes hydrolyzates were used as carbon and nitrogen source, potato starch, and rice bran hydrolyzates were the best carbon and nitrogen source, respectively. Key words: Kojic acid, Aspergillus flavus, mutation, UV, Gamma radiatio

Kinetics, Modelling and Scaling-Up of Kojic Acid Fermentation by Aspergillus Flavus 44-1 Using Different Carbon Sources

Kojic acid production by Aspergillus flavus strain 44- 1 using different types of carbon source (glucose, starch hydrolysate from enzymatic hydrolysis of sago starch, sucrose, fructose and gelatinized sago starch) was carried out in 250 mL shake flask, 2 L, 8 L and 50 L stirred tank fermenters. The experimental data from batch fermentation and resuspended cell system were analysed in order to form the basis for a kinetic model of the process. Unstructured model based on logistic and Luedeking-Piret equations was found suitable to describe growth, substrate consumption and kojic acid production by Aspergillus flavus in batch and also resuspended cell system using either glucose or sucrose. From the modelling, it was found that kojic acid production by A. flavus was non-growth associated process. The kinetic parameter values for each fermenter were calculated from the modelling and they can be used to verify the experimental data using various types and concentration of carbon source. Kojic acid production (23.5 g/l) using 100 g/L sago starch in a shake flask was comparable to fermentation of glucose (32.5 g/L) and starch hydrolysate (27.9 g/L) but in the 8 L and 50 L fermenter kojic acid production was greatly reduced due to non-optimal aeration conditions. Fed-batch fermentation with intermittent feeding of concentrated sago starch (140 g/L) can be employed to improve direct fermentation of sago starch to kojic acid by about 4 times higher as compared to batch fermentation. A. flavus was also capable to utilise sucrose for kojic acid fermentation where the highest production (40.23 g/L) in 2 L fermenter was obtained at 150 g/L sucrose. Kojic acid production (10.25 g/L) was greatly reduced in fermentation using fructose as the sole carbon source. Scaling-up based on a constant impeller tip speed (1.65 m/s) together with optimal DOT and pH control strategies was successfully used for kojic acid fermentation in 50 L fermenter using glucose and sucrose as carbon sources. Kojic acid fermentation by A. flavus can be divided into two phases; growth and production phase. The culture pH during growth phase influenced the performance of kojic acid fermentation to a further extend than did the pH during the production phase. The fermentation without pH controlled (started with an initial culture pH 3) showed higher kojic acid production than single-phase pH controlled fermentation at a range of pH 2.2 - 4.0. Comparable kojic acid production to fermentation without pH controlled was obtained in two-phase pHcontrol led fermentation (started with initial culture pH, without control during growth phase and switched to 3 during production phase). Efficient conversion of glucose to kojic acid was achieved in a resuspended cell system, in a solution containing only glucose with citrate buffer at pH 3.5 and 3 0°C. The rate of glucose conversion to kojic acid was increased with increasing glucose concentration up to 100 g/L, suggesting that the biotransformation of glucose to kojic acid by the cell-bound enzymes fol lowed the Michaelis-Menten enzyme kinetic models. The value of Km and Vmax for the reaction, as determined by using Langmuir plot, was 10.042 g/L glucose and 0.076 g/L.h, respectively

Optimization of Lipase-Catalysed Synthesis and Characterization of Kojic Acid Esters

Kojic acid ester was successfully synthesized by esterification using lipase from Pseudomanas cepacia (Amano PS) as a biocatalyst in an organic media. The reaction mixture consisted of 90 mM of oleic acid, 360 mM of kojic acid and 0.15 g Amano PS in the presence of 2 mL of acetonitrile. The mixture was incubated at temperature of 50°C for 24 hour at shaking speed of 150 rpm. Lipozyme IM, Novozym 435, Pseudomonas cepacia lipase, Aspergillus niger, Candida rugosa and Eupergit C-Lipase were tested for their suitability as the enzyme in the reaction. Among the enzymes tested, lipase from Pseudomonas cepacia gave the highest enzyme activity (0.015 yield/μg protein content) and specific activity (3.738 x 10-6 mmol ester/min/mg protein content) in the synthesis. Oleic acid was found to be the best substrate to produce the ester but must be in the presence of acetonitrile as an organic solvent. The maximum percentage yield using Pseudomonas cepacia lipase at optimal condition was 44% without removal of water from the reaction mixture. From the optimization studies, kojic acid derivatives can be synthesized from palm oil and kojic acid, by Pseudomonas cepacia lipase (Amano PS). TLC analysis showed that kojic acid ester gave an Rf value of 0.28 in the TLC plate and a retention time of 31.752 min in the gas chromatogram. The product formation and the reactant disappearance were monitored by IR spectroscopy. A strong C=O stretching of the ester at 1746 cm-1 and weak absorption peak of O-H in the unreacted kojic acid indicated completion of the esterification reaction. The gas chromatography-mass spectrometry (GC-MS) analysis gave a molecular ion peak at m/z 478 which was due to the kojic acid ester. 1H-NMR and 13C-NMR spectral data confirmed the molecular structure of the kojic acid ester

Optimization of kojic acid production conditions from cane molasses using Plackett-Burman design

Fungal synthesis of kojic acid has gained more interest in these days as an alternative way to chemical synthetic. The aspect of the microbial fermentation process is to develop a suitable culture medium to obtain the maximum amount of kojic acid using statistical methods. In this study; different selected three isolates of Aspergillus flavus (No 1, 2 and 3) were screened for their ability to produced kojic acid and the isolate No 3 was the highest kojic acid producer one. The capability of A. flavus No 3 to produce kojic acid was improved using Plackett-Burman design. From ten different agro-industrial wastes cane molasses recorded the highest kojic acid productivity with 2.24 g/l-1 day-1 and was the most effective parameter plays a crucial role in Plackett-Burman design. Maximum kojic acid production (24.65 g/l) by A. flavus (No. 3) obtained under the fermentation conditions: incubation temperature at 25oC, incubation time 9 days, pH 3, inoculum size 0.5%, shaking rate at 150 rpm and medium constituents: Cane molasses 60 g/l, yeast extract 7 g/l, KH2PO4 2 g/l, ZnSO4·7H2O 100 µg/l and MgSO4·7H2O 1 g/l with regression analysis (R2) 99.45% and 2.33-fold increase in comparison to the production of the original level (10.6 g/l). DOI: http://dx.doi.org/10.5281/zenodo.121151

Optimization of enzymatic synthesis of palm-based kojic acid ester using response surface methodology

Kojic acid monooleate is a fatty acid derivative of kojic acid which can be widely used as a skin whitening agent in a cosmetic applications. In avoiding any possible harmful effects from chemically synthesized product, the enzymatic synthesis appears to be the best way to satisfy the consumer demand nowadays. The ability of immobilized lipase from Rhizomucor meihei (lipozyme RMIM) to catalyze the direct esterification of kojic acid and oleic acid was investigated. Response Surface Methodology (RSM) and 5-level-4-factor central composite rotatable were employed to evaluate the effects of synthesis parameters such as enzyme amount (0.1-0.4 g), temperature (30-60 degrees C), substrate molar ratio (1-4 mmol, kojic acid:oleic acid) and reaction time (24-48 h) on percentage molar conversion to kojic acid monooleate. Analysis of the product using TLC, GC and FTIR showed the presence of kojic acid monooleate. The optimal conditions for the enzymatic reaction were obtained after analysis with backward elimination using 0.17 g of enzyme and 4 mmol of substrate at 52.50 degrees C for 42 h. Under these conditions the esterification percentage was 37.21%. The results demonstrated that response surface methodology can be applied effectively to optimize the lipase-catalysed synthesis of kojic acid monooleate. The optimum conditions can be used to scale up the process

The study of kojic acid as a chelating agent for cadium analysis

The absorption spectra of kojic acid and of cadmium kojate in various solvents have been obtained. From this data, the suitability of kojic acid as a possible analytical reagent for the determination of very small amounts of cadmium in water using ultraviolet absorption methods is discussed. Kojic acid is found to be not suitable for analysis of cadmium using ultraviolet absorption methods

Management of epidermal hyperpigmentation with a novel depigmenting formulation: a research survey

Background: Epidermal hyperpigmentation is an important dermatological concern with a high prevalence in the Indian population. Kojic acid-based depigmenting formulations have proven to be effective in the management of epidermal hyperpigmentation. A questionnaire-based survey was conducted to assess physicians’ knowledge about epidermal hyperpigmentation and practice patterns about a novel depigmenting formulation containing a combination of kojic acid dipalmitate 4%, azelaic acid 12%, glycolic acid 3%, niacinamide 4%, arbutin 2%, glycogen liquid 2%, sodium hyaluronate solution 2%, and shea butter 1% for epidermal hyperpigmentation treatment in India. Methods: The survey was conducted among 235 dermatologists across different geographical regions of India over 3 months. The questionnaire evaluated prevalence and choice and duration of therapies for epidermal hyperpigmentation in Indian clinical practice, and dermatologists’ real-life experience with efficacy of the novel formulation. Descriptive statistics were used to summarize survey results. Results: All 235 dermatologists completed the questionnaire and responded to questions based on experience in treating patients with epidermal hyperpigmentation. In all, 58% dermatologists preferred kojic acid-based combinations. Furthermore, 57% of dermatologists agreed that glycolic acid can enhance the penetration of kojic acid and azelaic acid, 47.45% strongly agreed that a kojic acid-based formulation could be a safe alternative to steroid and hydroquinone-based formulations. Overall, 67% of dermatologists agreed that the kojic acid-based formulations with additional moisturizers helped in faster resolution of epidermal hyperpigmentation compared to other depigmenting formulations. Conclusions: The survey findings indicate that the novel kojic acid-based formulation with additional moisturizers could be a preferred choice for epidermal hyperpigmentation management

Assessment of response of microdermabrasion with 2% kojic acid in melasma

Background: Melasma is an acquired disorder of hypermelanosis and several therapeutic modalities is in use to treat melasma. Kojic acid is a popular depigmenting agent, but its hydrophilic nature limits its transepidermal penetration. Microdermabrasion has been reported to increase the penetration of topical preparations. The objective of the study was to compare the efficacy of daily kojic acid (2%) gel along with biweekly microdermabrasion versus daily kojic acid (2%) gel alone in Indian patients.Methods: This study was carried out in 60 patients with melasma which were randomised into 2 groups of 30 patients each. The group I patients were treated with kojic acid 2% gel along with biweekly microdermabrasion, and group II patients were treated with kojic acid 2% gel alone. The results were assessed and compared after 12 weeks.Results: Both objective assessment and subjective assessment did not reveal a statistically significant difference in the treatment efficacy of two groups. However, the patients who received biweekly microdermabrasion were significantly more satisfied than those who did not.Conclusions: Microdermabrasion does not appear to have any synergistic effect with kojic acid in decreasing pigmentation in melasma. However, it does have a placebo effect on the patient.