Statistical optimization of kojic acid production by a UV-induced mutant strain of Aspergillus terreus

ABSTRACT The ability of four Aspergillus strains for biosynthesis of kojic acid was evaluated among which Aspergillus terreus represented the highest level (2.21 g/L) of kojic acid production. Improvement kojic acid production ability of A. terreus by random mutagenesis using different exposure time to ultraviolet light (5-40 min) was then performed to obtain a suitable mutant of kojic acid production (designated as C5-10, 7.63 g/L). Thereafter, design of experiment protocol was employed to find medium components (glucose, yeast extract, KH2PO4 (NH4)2SO4, and pH) influences on kojic acid production by the C5-10 mutant. A 25-1 fractional factorial design augmented to central composite design showed that glucose, yeast extract, and KH2PO4 were the most considerable factors within the tested levels (p < 0.05). The optimum medium composition for the kojic acid production by the C5-10 mutant was found to be glucose, 98.4 g/L; yeast extract, 1.0 g/L; and KH2PO4, 10.3 mM which was theoretically able to produce 120.2 g/L of kojic acid based on the obtained response surface model for medium optimization. Using these medium compositions an experimental maximum Kojic acid production (109.0 ± 10 g/L) was acquired which verified the efficiency of the applied method.

Antimicrobial activity of kojic acid from endophytic fungus Colletotrichum gloeosporioides isolated from Sonneratia apetala, a mangrove plant of the Sundarbans

Objective: To isolate and evaluate the antimicrobial activity of the active principle(s) from the ethyl acetate (EtOAc) extract of endophytic fungus Colletotrichum gloeosporioides (C. gloeosporioides) isolated from Sonneratia apetala. Methods: Water agar technique was used to isolate the fungus, and both microscopic and molecular techniques were used for identification of the strain. Potato dextrose broth was used to grow the fungus in large-scale. Reversed-phase preparative HPLC analysis was performed to isolate the major active compound, kojic acid. The EtOAc extract and kojic acid were screened for their antimicrobial activity against two Gram-positive and two Gram-negative bacteria as well as a fungal strain using the resazurin 96-well microtitre plate antimicrobial assay. Results: The fungus C. gloeosporioides was isolated from the leaves of Sonneratia apetala. Initial identification of the fugal isolate was carried out using spore characteristics observed under the microscope. Subsequently, the ITS1-5.8S-ITS2 sequencing was employed for species-level identification of the fungus C. gloeosporioides. Five litres of liquid culture of the fungus produced approximately 610 mg of a mixture of secondary metabolites. Kojic acid (1) was isolated as the main secondary metabolite present in the fungal extract, and the structure was confirmed by 1D, 2D NMR and mass spectrometry. The EtOAc extract and compound 1 exhibited considerable antimicrobial activity against all tested microorganisms. Whilst the minimum inhibitory concentration (MIC) values from the EtOAc extract ranged between 2.4× 10

Antimicrobial activity of kojic acid from endophytic fungus Colletotrichum gloeosporioides isolated from Sonneratia apetala, a mangrove plant of the Sundarbans

Objective:To isolate and evaluate the antimicrobial activity of the active principle(s) from the ethyl acetate (EtOAc) extract of endophytic fungus Colletotrichum gloeosporioides (C. gloeosporioides) isolated from Sonneratia apetala.Methods:Water agar technique was used to isolate the fungus, and both microscopic and molecular techniques were used for identification of the strain. Potato dextrose broth was used to grow the fungus in large-scale. Reversed-phase preparative HPLC analysis was performed to isolate the major active compound, kojic acid. The EtOAc extract and kojic acid were screened for their antimicrobial activity against two Gram-positive and two Gram-negative bacteria as well as a fungal strain using the resazurin 96-well microtitre plate antimicrobial assay.Results:The fungus C. gloeosporioides was isolated from the leaves of Sonneratia apetala. Initial identification of the fugal isolate was carried out using spore characteristics observed under the microscope. Subsequently, the ITS1-5.8S-ITS2 sequencing was employed for species-level identification of the fungus C. gloeosporioides. Five litres of liquid culture of the fungus produced approximately 610 mg of a mixture of secondary metabolites. Kojic acid (1) was isolated as the main secondary metabolite present in the fungal extract, and the structure was confirmed by 1D, 2D NMR and mass spectrometry. The EtOAc extract and compound 1 exhibited considerable antimicrobial activity against all tested microorganisms. Whilst the minimum inhibitory concentration (MIC) values from the EtOAc extract ranged between 2.4× 10Conclusions:The results revealed that the endophytic fungus C. gloeosporioides could be a good source of commercially important kojic acid, which exhibited antimicrobial properties.

Transdermal delivery of kojic acid from microemulgel.

The aim of this study was to develop microemulgel for skin delivery of kojic acid. Microemulsions (ME) containing either oleic acid (OA) or caprylic/capric triglycerides as the basic oily components were developed after construction of pseudoternary phase diagrams. Tween 80 was used as surfactant for oleic acid system both in presence and absence of ethanol or propylene glycol (PG) as cosurfactants. For the caprylic/capric systems, Tween 85 was the surfactant in presence or absence of ethanol as cosurfactant. Selected ME formulations were tested for transdermal delivery of kojic acid both in fluid state and after transformation into gel. Incorporation of cosurfactants expanded the microemulsion zone. The cosurfactant free ME were more viscous. Incorporation of kojic acid in ME systems increased the transdermal flux compared to saturated aqueous solution. caprylic/capric ME were more efficient than (OA) based ME. Transformation of the tested ME systems into gel produced significant enhancement in transdermal drug delivery compared with the saturated aqueous drug solution. However, the data revealed superior efficacy for the fluid ME systems over the corresponding microemulgel. In conclusion, both (OA) and caprylic/capric ME were promising for dermal and transdermal delivery of kojic acid even after gel formation.

Kojic Acid Peptide: A New Compound with Anti-Tyrosinase Potential

BACKGROUND: Kojic acid was used for decades in the cosmetic industry as an antimelanogenic agent. However, there are two major drawbacks of Kojic acid, one is cytotoxicity and second are instability on storage. These limitations led the scientist to synthesize the active Kojic acid peptides. OBJECTIVE: In the present study, we synthesize and investigate the effect of five Kojic acid peptides to overcome the limitation of Kojic acid. METHODS: The peptide was analyzed and purified by high-performance liquid chromatography and matrix-assisted laser desorption ionization time of flight mass spectroscopy. Further, the tyrosinase activities of the Kojic acid and Kojic acid peptides were compared. The toxicity was measured and the melanin content is recorded in B16F10 mouse melanoma cells. RESULTS: Maximum tyrosinase activity was measured by Kojic acid peptides. Therefore, Kojic acid peptides were subjected to melanin assay and cytotoxicity assay and finally the stability of the Kojic acid peptide was measured. CONCLUSION: It was observed that this newly synthesized Kojic acid peptide is stable and potent to inhibit the tyrosinase activity and melanin content of B16F10 mouse melanoma cells without exhibiting cell toxicity. Together, these preliminary results suggest that a further exploration is being needed to establish Kojic acid peptide as antimelanogenic agent.

Production of Some Polysaccharides from Kojic Acid Producing Aspergillus oryzae var effusus NRC14 Biomass.

Aims: The present investigation describes the utilization of Aspergillus oryzae var. effusus NRC14 biomass producing kojic acid in the production of fine chemicals that have valuable applications such as chitin, chitosan, β-1, 3 glucan and /or their derivatives. Methodology: Experiments of cell growth and substrate utilization were conducted in static cultures with identified medium composition. The technology involved in the extraction of their biopolymers is quite simple and the size scale is compatible with those currently used. This work was performed in Department of Microbial Chemistry, NRC. Results and Conclusion: The utilization of A. oryzae var. effusus NRC14 biomass waste actually will cover a part of the cost production of kojic acid; therefore, clean cell walls preparations were isolated from the fungus biomass at different stages during kojic acid fermentation in a glucose salts medium. The major chitin constituent was extracted from the prepared cell walls from six days cultured A. oryzae var. effusus NRC14 biomass by cold conc. HCl or by 5% of LiCl/ DMAA solvent in yields 4.78% and 2.44%, respectively. Among the chitin family, chitosan has received special attention since it has several applications. Chitosan was extracted from alkali insoluble material (cell wall) obtained from 12 days cultured biomass by using different concentrations of acetic acid (0.5-2.5%) the results showed that the highest yield of chitosan was 4.65% at a concentration of 2% acetic acid. The isolated product was characterized by infrared spectrum. Chitosanglucan complex was isolated in 17.94% yield by treating the A. oryzae var. effusus NRC14 biomass produced from kojic acid production for 12 days with 40% aqueous NaOH for 6h. at 95°C. Chitosan-glucan complex was fractionated into its components. The amount of extracted chitosan was 3.5%; the residue obtained after solubilization of chitosan by acid treatment was characterized as being mostly insoluble glucan (94%). Chitin-glucan complex was isolated from 6 days cultured A. oryzae var. effusus NRC14 biomass in yield 17.034%. The lyophilized product was fractionated into its components (chitin and glucan) in yields 16.36% chitin, 82.72% β, 1-3 glucan. The infrared spectra of the isolated products as well as of standard samples proved the identity of the isolated materials as chitosan, chitin and glucan.

Effect of kojic acid on immune systems of mice damaged by gamma-irradiation / 军事医学

Objective To evaluate the effect of kojic acid( KA) on the immune system of mice after exposure to gam-ma-irradiation.Methods Twenty male C57BL/6 mice were divided into normal group, irradiation group, low/high doses of KA pretreated groups.Mice in normal group did not receive any treatment,while mice in other groups were sc injected with a single dose of sterile distilled water or KA(75 and 300 mg/kg, respectively) 27 h prior to a sublethal dose(4 Gy, 138.54 and 140.30 cGy/min, respectively) of whole body gamma-irradiation.The injected volume was calculated by 0.2 ml/20 g.Forty mice were sacrificed at day 2 and day 8 post-irradiation, respectively.The splenic lymphocyte transformation and spleen and thymus indexes were determined.Histopathological sections were produced, and the morphological changes were also observed.Results The splenic lymphocyte transformation capacity and spleen and thymus indexes of mice pre-treated with 300 mg/kg elatve mass KA were increased significantly ( P<0.01) compared with the irradiation group.The morphological changes in the spleen and thymus of mice in 300 mg/kg KA pretreated group were better than in the irradia-tion group.The above parameters of mice in irradiation group were injured severely in comparison with the normal group. Conclusion Acute radiation can damage the immune system of mice obviously.KA can enhance the transformation capaci-ty of lymphocytes and has marked protective effect on the immune system of mice after irradiation.

Chemical constituents in flowers of Rhododendron lapponicum / 中草药

Objective: To study the chemical constituents in the flowers of Rhododenron parvifolium, which is a traditional Tibet medicine. Methods: Silica gel column chromatography, HPLC, and Sephadex LH-20 column chromatography techniques were used for the separation and purification of the compounds. Their structures were determined on the basis of IR, MS, 1D, and 2D NMR spectral evidences. Results: Thirteen compounds were isolated from methanol extract in the flowers of R. parvifolium, and their structures were identified as parvifoliumol I (1), umbelliferone (2), scopoletin (3), 4', 5, 7-trihydroxyflavanone (4), 8-demethylfarrerol (5), farrerol (6), quercetin (7), rutin (8), eudesmin A (9), 4-hydroxybenzoic acid (10), 3, 5-dihydroxy toluene (11), oleanane (12), and kojic acid (13). Conclusion: Compound 1 is a new compound, which has a hexahydroxanthene skeleton. Compounds 9, 11, and 13 are obtained from the plants of Rhododendron L. for the first time.

Modulation of antimicrobial metabolites production by the fungus Aspergillus parasiticus

Biosynthesis of active secondary metabolites by fungi occurs as a specific response to the different growing environments. Changes in this environment alter the chemical and biological profiles leading to metabolites diversification and consequently to novel pharmacological applications. In this work, it was studied the influence of three parameters (fermentation length, medium composition and aeration) in the biosyntheses of antimicrobial metabolites by the fungus Aspergillus parasiticus in 10 distinct fermentation periods. Metabolism modulation in two culturing media, CYA and YES was evaluated by a 2² full factorial planning (ANOVA) and on a 2³ factorial planning, role of aeration, medium composition and carbohydrate concentration were also evaluated. In overall, 120 different extracts were prepared, their HPLC profiles were obtained and the antimicrobial activity against A. flavus, C. albicans, E. coli and S. aureus of all extracts was evaluated by microdilution bioassay. Yield of kojic acid, a fine chemical produced by the fungus A. parasiticus was determined in all extracts. Statistical analyses pointed thirteen conditions able to modulate the production of bioactive metabolites by A. parasiticus. Effect of carbon source in metabolites diversification was significant as shown by the changes in the HPLC profiles of the extracts. Most of the extracts presented inhibition rates higher than that of kojic acid as for the extract obtained after 6 days of fermentation in YES medium under stirring. Kojic acid was not the only metabolite responsible for the activity since some highly active extracts showed to possess low amounts of this compound, as determined by HPLC.

Efficacy and Safety of Hydroquinone, Kojic Acid and Glycolic Acid Combination in the Treatment of Melasma.

Background & Methodology: It was an opened clinical trial. The study was carried out from March 2009 to February 2011, in the outpatient department of Dermatology and Venereology, Bangabandhu Sheikh Mujib Medical University (BSMMU) Dhaka, Bangladesh to evaluate the efficacy and side effects of the combination therapy of hydroquinone(HQ), kojic acid(KA) and glycolic acid(GA) for the treatment of melasma. Patients suffering from melasma were selected as study population. Within the period of data collection, thirty patients of melasma were assigned purposively. The efficacy was evaluated using Melasma Area and Severity Index (MASI) Score. The severity of melasma of each of the four regions (forehead, right malar region, left malar region and chin) are assessed based on three variables, percentage of the total area involved (A), darkness (D) and homogenicity (H). Results:The study showed that majority (30%) of the cases were between 20 to 25 years, majority (77%) of patients were female, (50%) patients were housewives, (50%) were in graduation level of education, (50%) of patients was found as upper class and negative family history was present in majority (80%) of cases. It was seen that highest (93%) number of patients were malar type and 7% were centro-facial type. The study showed the change in MASI Score after treatment with combination therapy (GA 2%, HQ 2% and KA 1%). After 12 weeks of treatment, the average MASI score was decreased by 24.20% indicating mild reduction of the severity of melasma and 18%, 18%, 9% and 55% patients developed side -effects like itching, burning sensation, scaling, and erythema respectively. Conclusion: Combination therapy (GA 2%, HQ 2% and KA 1%) has a few lightening effect on melasma, with no remarkable side effects. Further Further study should be conducted with large number of sample and longer follow up.

Biotransformation of sucrose into 5-hydroxy-2-hydroxymethyl-γ-pirone by Aspergillus flavus

The sucrose hydrolysis and the preference of consumption of glucose instead of fructose were investigated for the production of 5-hydroxy-2-hydroxymethyl-γ-pyrone (HHMP) in the presence of Aspergillus flavus IOC 3974 cultivated in liquid Czapeck medium. Standardized 0.5g of pellets were transferred as inoculum into twelve conical flasks of 250 ml containing 100 ml of medium with different sucrose concentration, which was kept at 120 rpm and 28"C for 16 days without pH adjustment. Aliquots of 500μl of the broth culture were withdrawn at 24 h intervals and analyzed. The major yield of HHMP was 26g l-1 in 120g l-1 of sucrose. At these conditions, A. flavus produced an invertase capable of hydrolyzing 65 percent of total sucrose concentration in 24h, and an isomerase capable of converting fructose into glucose. In this work, it focused the preference for glucose and, then, of fructose by A. flavus and the strategy used to produce HHMP.

Foram investigadas a hidrólise da sacarose e a preferência pela glicose frente à frutose no processo de produção do 5-hidroxi-2-hidroximetil-γ-pirona (HHMP) na presença de Aspergillus flavus IOC 3974 cultivado em meio líquido Czapeck. Quantidades de 0,5g de pelletes foram utilizadas como inóculo. Doze frascos cônicos de 250 ml contendo 100 ml de meio de culturacom diferentes concentrações de sacarose foram utilizados.Os microrganismos foram cultivados a 120 rpm e 28"C por 16 dias sem ajuste do pH. O maior rendimento do HHMP foi 26g l-1em 120g l-1de sacarose. Nestas condições, A. flavus, foi capaz de produzir uma invertase possibilitando a hidrólise de 65 por cento da concentração total de sacarose em 24 horas, conjuntamente com a produção de uma isomerase que foi capaz de converter a frutose em glicose. Este trabalho está focalizado preferencialmente no consumo da glicose frente à frutose por A. flavus e na estratégia de produção do HHMP.

In vitro evaluation of copaiba oil as a kojic acid skin enhancer

The capacity of copaíba oil to act as a skin penetration enhancer for the depigmenting agent kojic acid was evaluated using an in vitro diffusion system with static flux and shed rattlesnake skin membrane, Crotalus durissus terrificus, in saline solution at 34±2 ºC as the fluid receptor. The quantities of kojic acid liberated into the fluid receptor were determined by spectrophotometry at 268 nm with intervals of one and a half hours. The membranes, pretreated with copaíba oil at 25 percent and 50 percent v/v, gave flux values of 8.0 and 12.7 µg/cm²/h, permeability values of 2.0 and 3.3 cm×10-4/h, and promotion factors of 4.1 and 3.7, respectively. These results indicate that copaíba oil, at the two concentrations studied, has the capacity to promote penetration of kojic acid.

A propriedade do óleo de copaíba como agente promotor de penetração cutânea do despigmentante ácido kójico foi avaliada utilizando-se sistema de difusão in vitro com fluxo estático, membrana de pele da serpente cascavel - Crotalus durissus terrificus e solução salina a 34±2 ºC como fluido receptor. As quantidades liberadas do ácido kójico no fluido receptor foram determinadas por espectrofotometria em 268 nm em intervalos de 1:30 h. As membranas pré-tratadas com óleo de copaíba a 25 e 50 por cento v/v apresentaram valores de fluxo de 8,0 e 12,7 µg/cm²/h, permeabilidade de 2,0 e 3,3 cm×10-4/h, e fatores de promoção de 4,1 e 3,7, respectivamente. Os resultados obtidos indicaram que o óleo de copaíba, nas duas concentrações estudadas, apresentou capacidade de promoção da penetração do ácido kójico.

Permeação cutânea in vitro do ácido kójico / Kojic acid in vitro percutaneous penetration study

Desordens no processo melanogênico podem causar as hiperpigmentações, sendo a de maior freqüência o melasma. O ácido kójico é um dos despigmentantes tópicos utilizados no tratamento destas hipercromias. A ação de um produto dermatológico deve ser tópica, não devendo atingir níveis sistêmicos. Um dos fatores que pode definir o nível de ação, tópica ou sistêmica, são os excipientes das formulações. Neste contexto, o presente trabalho teve por objetivo avaliar o grau de permeação cutânea in vitro, utilizando célula de FRANZ modificada, de uma formulação em relação a uma solução tampão pH 7,4, ambas contendo ácido kójico na concentração de 2 por cento. A quantificação do ácido kójico permeado e retido na membrana foi realizada por método espectrofotométrico no ultravioleta. O estudo in vitro mostrou que a absorção do ácido kójico da formulação apresentou cinética de pseudo 1ª ordem (no intervalo de 1 a 8 horas) e, conseqüentemente, menor fluxo através da membrana natural (pele da orelha de porco) e maior retenção cutânea, enquanto que o ácido kójico da solução tampão pH 7,4 apresentou cinética de ordem zero e, conseqüentemente, maior fluxo e menor retenção. Este resultado indicou que a formulação desenvolvida mostrou-se adequada para a veiculação do ácido kójico, tendo em vista que o órgão alvo é a pele.

Disorder of the tirosinase biosynthesis process can result on hiperpigmentations, like the frequently found melasma. The kojic acid is one of the depigmenting topic agent utilized in the handling of these hipercromies. The action of a dermatologic product should be topic, should not reach systemic levels. One of the factors that is able to defined the level of action, topic or systemic, is the vehicle. In this contest, the present work had for objective evaluate the rank of in vitro cutaneous permeation, in the cell of FRANZ modified, of a formulation regarding a buffered solution 7.4, both containing kojic acid (2 percent). The kojic acid percutaneous penetration and retention was quantified by a spectrophotometric in the ultraviolet method. The in vitro study showed that the absorption of kojic acid from the formulation presented kinetic of pseudo 1st order (in the break from 1 to 8 hours) and consequently smaller stream through the natural membrane (skin from the ear of pig) and bigger cutaneous retention, while the kojic acid from the buffered solution pH 7.4 presented kinetic of zero order and consequently bigger stream and smaller retention. This result indicated that the formulation developed can be used as a vehicle for kojic acid, having in mind that the target tissue is the skin.

Fungistatic Activity of Kojic Acid Against Human Pathogenic Fungi and Inhibition of Melanin-production in Cryptococcus neoformans

Kojic acid was investigated for its antifungal activity against the human pathogenic fungi including Candida albicans, Cryptococcus neoformans and Trichophyton rubrum. For C. albicans, C. neoformans and T. rubrum, the MIC (minimum inhibitory concentration) of kojic acid was 640, 80 and 160 microg/ml, respectively. In C. neoformans, melanin-producing yeast, kojic acid-treated nonmelanized cell was more susceptible to magainin than melanized cell, suggesting melanin give a protective function against microbial peptide.

COMPOUND MUTATION BREEDING OF KOJIC ACID PRODUCTION STAIN / 微生物学通报

mutant (UCN 7 17) of producing high yield Kojic acid was screened fr om Aspergillus flavus after treated with UV three times, ? ray of 60 Co one time and NTG four times, underoptimal conditions, the Kojic aci d production level reached up to 6 3% after 7 days, compared with original stains 0 926% The experiments showed that compound mutation using various mutagenic agents ca n alter the original stains sensitivity to mutagenic agents, increase mutation frequency and raise Kojic acid yield

Metabolic Regulation of Cell Growth and Antimicrobial Metaboliteof the F0238 / 微生物学通报

Metabolic regulation of cell growth and antimicrobial metabolite of the F0238 was carried out in this study.The proper compositions of the culture medium and technological conditions for its fermentation were investigated.The results showed that the optimum conditions for the growth of the strain and its production of kojic acid were PDA medium with starch 2% as C source, peptone 0.2% as N source, and temperature 28℃, culture time 5 day (144 h), culture volume 80mL/500mL Erlenmeyer flask. Under above conditions, the dynamic curve of fermentation was obtained by an automatic mini-bioreactor of 10L and indicated a trend of decreasing pH, DO and residual sugar and increasing biomass and kojic acid production by F0238.