The primary molecular target of Kojic acid is tyrosinase, a copper-containing enzyme that catalyzes the rate-limiting step in melanin biosynthesis: the hydroxylation of tyrosine to DOPA and the subsequent oxidation of DOPA to DOPAquinone. Kojic acid acts as a competitive inhibitor by chelating the copper ions at the enzyme's active site, thereby blocking the catalytic activity of tyrosinase. The IC50 value for mushroom tyrosinase is approximately 30.6 μM. In addition to tyrosinase, Kojic acid also inhibits other copper-dependent enzymes, such as xanthine oxidase and certain amino acid oxidases. It has been shown to inhibit NF-κB activity and downregulate UVR-induced NF-κB expression in human keratinocytes.

In vitro studies have demonstrated that Kojic acid effectively inhibits tyrosinase activity in a dose-dependent manner. The IC50 for mushroom tyrosinase is approximately 30.6 μM. At a concentration of 50 μg/mL, Kojic acid was found to decrease cell growth by 62% (IC50 34 μg/mL) and by 79% (IC50 27.84 μg/mL) in certain cell lines. It also exhibits anti-leptospira effects with a minimum inhibitory concentration (MIC) of 1-10 μg/mL. In human keratinocyte transfectants, Kojic acid inhibits cellular NF-κB activity and downregulates ultraviolet radiation (UVR)-induced activation of NF-κB expression. Its antioxidant activity is attributed to its ability to scavenge free radicals and chelate metal ions.

In vivo studies have evaluated the skin-lightening and anti-inflammatory effects of Kojic acid in animal models. When applied topically to the skin of guinea pigs or mice, Kojic acid reduces melanin pigmentation in a dose-dependent manner, consistent with its tyrosinase inhibitory activity. Oral administration of Kojic acid has been shown to exhibit antioxidant and anti-inflammatory effects in rodent models of oxidative stress and inflammation. Its ability to penetrate the skin and reach melanocytes in the basal layer of the epidermis supports its efficacy as a topical depigmenting agent. However, the compound's stability and skin penetration can be enhanced through formulation with penetration enhancers or derivatives.

The non-cellular enzyme inhibition activity of Kojic acid can be assessed using purified mushroom tyrosinase in a cell-free assay. In a typical protocol, tyrosinase enzyme is incubated with L-DOPA or L-tyrosine as substrate in phosphate buffer, and varying concentrations of Kojic acid are added. The reaction progress is monitored spectrophotometrically by measuring the increase in absorbance at 475 nm, corresponding to the formation of DOPAchrome. The IC50 is calculated by plotting the initial reaction velocity versus the inhibitor concentration. This assay provides a direct measure of Kojic acid's ability to inhibit tyrosinase activity without cellular interference. Additional assays using xanthine oxidase can evaluate its inhibitory effects on other copper-dependent enzymes.

Cellular assays for Kojic acid typically employ melanocyte cell lines, such as B16 mouse melanoma cells or human melanocytes, to evaluate its effects on melanin synthesis. In a typical protocol, cells are cultured in appropriate medium and treated with varying concentrations of Kojic acid (ranging from 0 to 200 μM) for 48-72 hours. Melanin content is measured by solubilizing the cells in sodium hydroxide and measuring absorbance at 405 nm. Cell viability is assessed using MTT assay to distinguish between melanin inhibition and cytotoxicity. The inhibition of tyrosinase activity can also be confirmed by measuring intracellular tyrosinase activity in cell lysates. These assays demonstrate Kojic acid's dose-dependent reduction in melanin production.

In vivo animal experiments for Kojic acid typically involve topical application on the skin of guinea pigs, mice, or other pigmented animal models. In a standard protocol, Kojic acid is formulated in a suitable cream or gel and applied to a defined area of depilated skin once or twice daily for several weeks. Pigmentation is assessed visually and measured using a chromameter to quantify changes in skin color (L* value) over time. Skin biopsies may be collected for histological analysis to evaluate melanin content and melanocyte density. In models of UV-induced hyperpigmentation, animals are exposed to UV radiation before Kojic acid treatment to induce melanin production, and the compound's ability to reverse pigmentation is assessed.

Metabolism / Metabolites
The structure of kojic acid indicates that its metabolic pathway is relatively simple, similar to that of dietary hexoses. After being absorbed by the gastrointestinal tract, kojic acid enters the bloodstream, and its metabolic process may be similar to that of hexoses. (A3073)
The pharmacokinetic properties of Kojic acid include both oral and transdermal absorption. When applied topically, Kojic acid can penetrate the stratum corneum and reach the basal layer of the epidermis, where melanocytes are located. Its systemic absorption is limited, making it suitable for topical applications with minimal systemic side effects. Following oral administration, Kojic acid is absorbed from the gastrointestinal tract and distributed to various tissues. It is metabolized primarily in the liver and excreted via the kidneys. The compound's small molecular weight (142.11 g/mol) and hydrophilic nature facilitate its absorption and distribution. Its half-life and bioavailability support its use in cosmetic formulations for skin-lightening purposes.

Toxicity Summary
Kojic acid is a competitive and reversible inhibitor of animal and plant polyphenol oxidases (tyrosinase), xanthine oxidase, and D- and certain L-amino acid oxidases. Inhibition of tyrosinase prevents melanosis, while inhibition of oxidases prevents the metabolism of certain amino acids. Kojic acid also reversibly affects thyroid function by inhibiting iodine uptake, leading to decreased levels of thyroid hormones T3 and T4 and increased levels of thyroid-stimulating hormone (TSH). Increased TSH secretion from the pituitary gland, in turn, stimulates thyroid hyperplasia. (A3073, A3074) Interactions Kojic acid (5-hydroxy-2-hydroxymethyl-γ-pyranone) is a bacterial metabolite widely used in the food industry. In the presence of visible light and molecular oxygen, kojic acid can cause DNA breaks in calf thymus. The presence of transition metal ions Fe(III), Fe(II), and Cu(II) significantly enhances this degradation. DNA breaks in the presence of Fe(III) do not appear to have specific break sites or sequences. Kojic acid catalyzes the reduction of transition metals, with Cu(II) playing a crucial role in DNA degradation. Kojic acid can also reduce oxygen to superoxide anions and generate hydroxyl radicals in the presence of metal ions. The inhibitory effects of superoxide dismutase, catalase, iodides, mannitol, formate, and sodium azide on DNA breakage confirm the involvement of these reactive oxygen species in this reaction. ...The anti-wrinkle activity of kojic acid was evaluated using hairless mice exposed to chronic simulated ultraviolet (UV) sunlight as a model animal. After a 20-week exposure period, topical application of kojic acid before UV exposure was observed to significantly prevent: (1) wrinkles, (2) epidermal hyperplasia, (3) subdermal fibrosis, and (4) an increase in the extracellular matrix components of the upper dermis. These results suggest that kojic acid is a typical drug for preventing skin wrinkles caused by chronic photodamage. This study investigated the individual and combined effects of kojic acid and aflatoxin in male broiler chickens (Peterson x Hubbard). This experiment employed a 2×2 factorial design, with dietary treatments of 0 and 2500 mg/kg kojic acid and 0 and 2.5 mg/kg aflatoxin, respectively. Broilers were purchased at 1 day old and housed in electrically heated chicken houses with free access to feed and water until 3 weeks of age. Kojic acid poisoning was characterized by significantly decreased body weight, relative bursal weight, serum cholesterol concentration, and serum alkaline phosphatase activity (P<0.05), as well as significantly increased relative weight of the pancreas, proventriculus, and gizzard, and serum uric acid and triglyceride concentrations (P<0.05). Aflatoxin poisoning was characterized by significantly decreased body weight, serum total protein, albumin, cholesterol and inorganic phosphorus concentrations, serum aspartate aminotransferase activity, mean corpuscular volume, mean corpuscular hemoglobin content, and mean corpuscular hemoglobin concentration (P<0.05). Aflatoxin alone significantly (P<0.05) increased the relative weight of the liver, kidneys, spleen, pancreas, proventriculus, and heart, as well as serum pyruvate transaminase activity. The only significant (P<0.05) interaction between kojic acid and aflatoxin, described as an antagonistic effect, was an increase in mean corpuscular hemoglobin (MCH) content and concentration. These data suggest that, at the concentrations used in this study, kojic acid is not an synergist for aflatoxin.
The toxicological profile of Kojic acid has been extensively studied due to its widespread use in cosmetics and food. It is generally recognized as safe (GRAS) for topical application at concentrations up to 1-2%. Common side effects include mild skin irritation, redness, and contact dermatitis in sensitive individuals. At higher concentrations, Kojic acid may cause sensitization and allergic reactions. Oral toxicity studies have shown that Kojic acid has a low acute toxicity profile, with no significant genotoxic or carcinogenic effects at typical exposure levels. However, prolonged use at high concentrations should be monitored to prevent potential adverse effects. It is contraindicated in individuals with known hypersensitivity to the compound.

Kojic acid is a pyranone compound with a 4H-pyran ring, substituted at positions 5, 2, and 4 with hydroxyl, hydroxymethyl, and carbonyl groups, respectively. It has been isolated from Aspergillus oryzae. Kojic acid possesses various biological activities, including NF-κB inhibition, Aspergillus oryzae metabolite, skin whitening agent, catechol oxidase inhibitor (EC 1.10.3.1), laccase inhibitor (EC 1.10.3.2), quercetin 2,3-dioxygenase inhibitor (EC 1.13.11.24), tyrosinase inhibitor (EC 1.14.18.1), and D-amino acid oxidase inhibitor (EC 1.4.3.3). It is an enol, a primary alcohol, belonging to the 4-pyranone class of compounds. It is derived from the hydride of 4H-pyran. Kojic acid has been reported in Phaeosphaeria fuckelii, Aspergillus flavus, and several other organisms with relevant data. Kojic acid is a synthetic intermediate in the production of food additives. Studies have shown that kojic acid possesses antitumor activity (A7859). The mechanism of action of kojic acid is well-established; it has been shown to act as a competitive and reversible inhibitor of polyphenol oxidases, xanthine oxidases, and D- and some L-amino acid oxidases in plants and animals. In human HaCaT and SCC-13 cells transfected with kojic acid (a tyrosinase inhibitor that inhibits melanin biosynthesis in melanocytes)-induced NF-κB activation was investigated. This study used two keratinocyte cell lines transfected with the pNF-κB-SEAP-NPT plasmid to detect NF-κB activation. Transfected cells release secreted alkaline phosphatase (SEAP) as a transcriptional reporter gene upon NF-κB activation and contain the neomycin phosphotransferase (NPT) gene, a key selection marker for heritable mycin resistance. NF-κB activation was determined using a fluorescence detection method via the SEAP reporter gene. Kojic acid inhibited NF-κB activity in two human keratinocyte transfections. It also downregulated UVR-induced NF-κB expression activation in transfected HaCaT cells. Furthermore, kojic acid exhibited stronger inhibitory activity in transfected HaCaT cells than known antioxidants such as vitamin C and N-acetyl-L-cysteine. These results suggest that kojic acid is a potential inhibitor of NF-κB activation in human keratinocytes and hint that NF-κB activation may be involved in kojic acid-induced anti-melanogenesis effects.

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Therapeutic Uses
Depigmenting Agent/For Skin Whitening/
Chloasma is a chronic, relapsing condition. Due to the lack of effective treatments and the widespread perception that it is merely a cosmetic issue, melasma has been consistently underdiagnosed and undertreated. Hydroquinone, corticosteroids, licorice extract, and kojic acid have all been used as monotherapy for melasma. However, the current standard of care for melasma is combination therapy. To date, the most effective treatment is a triple therapy containing 4% hydroquinone, 0.05% retinoic acid, and 0.01% fluocinolone acetonide…
…Combination therapy, including frequent use of superficial and intermediate chemical peels, appears to be particularly effective and well-tolerated for patients with dark skin and melanosis. Post-inflammatory hyperpigmentation is the result of excessive pigment deposition following inflammatory skin conditions. The efficacy of topical retinoic acid, hydroquinone, azelaic acid, kojic acid, and glycolic acid peels varies…
Facial and neck pigmentation is common in middle-aged women and is associated with endogenous factors (hormones) and exogenous factors (such as the use of cosmetics and perfumes and exposure to sunlight). Melasma (also known as pregnancy mask) is the most common cause of facial pigmentation, but there are many other types, such as Riehl's melanosis, Sivater's heterochromia, Brock's perilipotic erythema pigmentosum, facial and neck follicular erythema melanosis, linea nigra, and cosmetic hyperpigmentation. Treatment of melasma and other facial pigmentation has always been challenging and frustrating… Various depigmenting agents have been tried with varying results. Topical application of 2% to 4% hydroquinone, or in combination with 0.05% to 0.1% retinoic acid, is a well-established treatment. Topical application of 15% to 20% azelaic acid is comparable in efficacy to hydroquinone but less irritating. Retinoic acid is particularly effective for treating hyperpigmentation in photoaged skin. Kojic acid, alone or in combination with glycolic acid or hydroquinone, has shown good efficacy due to its inhibitory effect on tyrosinase. Chemical peels can be used to treat melasma: trichloroacetic acid, Jessner solution, Una paste, alpha-hydroxy acid preparations, kojic acid, and salicylic acid, alone or in various combinations, have shown good efficacy. In contrast, laser therapies have not yet achieved completely satisfactory results, as they can induce hyperpigmentation and are prone to recurrence. In the future, new laser therapies promise to successfully remove facial hyperpigmentation. For more complete data on the therapeutic uses of kojic acid (7 types in total), please visit the HSDB record page.
Kojic acid is a naturally occurring fungal metabolite with a long history of use in the cosmetic industry as a skin-lightening and anti-aging agent. It is also employed as a food additive to prevent browning and as an antioxidant in various products. The compound exhibits multiple biological activities beyond tyrosinase inhibition, including antimicrobial, anti-inflammatory, and antioxidant properties. Kojic acid derivatives are being explored for their potential anti-tumor and anti-aging effects. It is available in various formulations, including creams, lotions, and serums, and is often combined with other depigmenting agents such as arbutin or hydroquinone for enhanced efficacy. Regulatory agencies worldwide have approved its use in cosmetic products at specified concentrations.

C6H6O4

142.1094

142.026

501-30-4

3840

Prismatic needles from acetone, ethanol+ether or methanol+ethyl acetate
Crystals
Prisms, needles from acetone

1.5±0.1 g/cm3

401.7±45.0 °C at 760 mmHg

152-155 °C(lit.)

179.9±22.2 °C

0.0±2.1 mmHg at 25°C

1.607

-0.64

2

4

1

10

214

0

O1C([H])=C(C(C([H])=C1C([H])([H])O[H])=O)O[H]

BEJNERDRQOWKJM-UHFFFAOYSA-N

InChI=1S/C6H6O4/c7-2-4-1-5(8)6(9)3-10-4/h1,3,7,9H,2H2

5-hydroxy-2-(hydroxymethyl)pyran-4-one

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ≥ 100 mg/mL (~703.68 mM)
H2O : ~50 mg/mL (~351.84 mM)

Solubility in Formulation 1: 5 mg/mL (35.18 mM) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with sonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 50.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (17.59 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 2.5 mg/mL (17.59 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 4: 14.29 mg/mL (100.56 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with ultrasonication (<60°C).

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 (Please use freshly prepared in vivo formulations for optimal results.)