Kojic Acid And Melasma

Author: Gabriele Fasano
Date: 14/07/2014




Melasma (a term derived from the Greek word ‘‘melas’’ meaning black) is a common acquired hypermelanosis that occurs exclusively in sunexposed areas, mostly in the face and occasionally in the neck and forearms (rarely).

Epidemiology and etiology

Melasma is more common in women. Men have been reported to represent 10% of cases [3] and demonstrate the same clinical and histologic characteristics as women [4]. The disease affects all racial groups, but there is a particular prominence among Hispanics and Asians [2]. Melasma is more apparent during and after periods of sun exposure. The exact cause of melasma remains elusive, but the two most important factors implicated in its etiopathogenesis are sunlight and genetic predisposition. Exacerbations of melasma are inevitably seen after a period of sun exposure and, conversely, melasma gradually fades during periods of sun avoidance. Genetic factors are also involved as suggested by familial occurrence and the higher prevalence of the disease among Hispanics and Asians. Other factors incriminated in the pathogenesis of melasma include pregnancy, oral contraceptives, estrogen-progesterone therapies, thyroid dysfunction, cosmetics, and phototoxic and antiseizure drugs [5].

Clinical features and classification

The number of hyperpigmented patches may range from a single lesion to multiple patches located usually symmetrically on the face and occasionally the V-neck area. The lesions have serrated, irregular, and geographic borders. According to the distribution of lesions, three clinical patterns of melasma are recognized: The centrofacial pattern (the most common), The malar pattern, The mandibular pattern.

Origin, Clinical Presentation, and Diagnosis of Facial Hypermelanoses


Melanogenesis is initiated with the first step of tyrosine oxidation by tyrosinase. When the skin is exposed to UV radiation, the formation of abnormal melanin pigment occurs, which constitutes a serious esthetic problem that is particularly prevalent in middle-aged and elderly individuals (3,4).
Tyrosinase (EC is a copper-containing enzyme that catalyzes two distinct reactions of melanin biosynthesis: the hydroxylation of tyrosine to 3,4-dihydroxy-phenylalanine (L-DOPA) by monophenolase action and the oxidation of L-DOPA to o-dopaquinone by diphenolase action. However, if L-DOPA is an active cofactor, its formation as an intermediate during o-dopaquinone production is still controversial. o- Dopaquinone is unstable in aqueous solution and rapidly undergoes a non-enzymatic cyclization to leukodopachrome, which is further oxidized non-enzymatically by another molecule of o-dopaquinone to yield dopachrome and one molecule of regenerated L-DOPA (5–7).
Tyrosinase exists widely in plants and animals tissues, and is involved in the formation of melanin pigments (8–10).

Synthesis and tyrosinase inhibitory properties of some novel derivatives of kojic acid


In China for many centuries, cereal grains, particularly rice, were converted to alcoholic beverages using a fermentation initiated by a fungus, that produces useful enzymes (e.g., amylases, proteinases), providing substrates for further yeast or bacterial fermentations. The fungal ‘starter’ was selected in 1121–220 BC. In the seventh century, Buddhist priests apparently brought their methods from China to Japan, where the fungal inoculum, often Aspergillus oryzae, became known as koji. After fungal growth on steamed rice, the resulting koji was used as a starter in brewing a rice wine (sak´e), or in making shoyu (soy sauce).
One hundred years ago, the koji process in Japan led to the isolation of the material now known as kojic acid. In 1912, Teijiro Yabuta described the process: portions of steamed rice (150 g) were inoculated with A. oryzae and kept at 30–35 ◦C for 2–3 weeks, with intermittent shaking, until fully covered with mycelium and spores. After low-temperature drying, the rice was finely powdered and ether-extracted in a Soxhlet apparatus. Yabuta applied the name kojic acid to the product, and obtained about 50 g of material from 15 kg of rice.
In the next years, further works showed that not only A. oryzae prodces Kojic Acid, but also other species of Aspergillus, like Flavus, Effusus, and some bacteria.
The apparently simple structural relationship between the pyranose ring structure of glucose and the pyrone ring structure of kojic acid had caught the attention of early investigators. For a direct conversion of glucose to kojic acid, one oxidation (CHOH →CO) and two dehydrations are required (not necessarily in that
order). On the other hand, kojic acid formation was observed with many substrates, pentoses, glycerol and dihydroxyacetone, ethanol, lactate, pyruvate, gluconic acid, mannitol, sorbitol.
Over the years, Kojic Acid has been used in many applications:
- BGYF reaction to detect A. flavus (and hence possible aflatoxin formation)
- Formation of pyridones
- Polymer formation
- Tyrosinase activity inhibition: discovered in 1979, this skill is due to the chelation of the copper component of this enzyme, also involved in the melanogenesis of the skin. Since this finidings Kojic Acid has been used to prevent melanosis in plant and seafood products, to increase the shelf-life of the products. This role has also been showed in frog epidermidis and in black goldfish. Moreover, kojic acid can suppress melaninogenesis, by inhibiting the PPO oxidation of L-Tyr and removing the o-quinones by combination, thus preventing polymerization to melanins.
In view of its inhibitory activity on tyrosinase and its inhibition of melaninogenesis, it was perhaps only natural that kojic acid came to be used as a skin lightening agent. This cosmetic use was approved by health authorities in Japan in 1988, to treat hypermelanosis like Melasma.

From miso, saké and shoyu to cosmetics: a century of science for kojic acid


Several methods of treatment are available to patients with melasma.

  1. First-line therapy usually consists of topical compounds that affect the pigment production pathway, broad-spectrum photoprotection, and camouflage.
  1. Second-line therapy often consists of the addition of chemical peels, although these must be used cautiously in patients with darker skin.
  1. Laser and light therapies represent potentially promising options for patients who are refractory to other modalities, but also carry a significant risk of worsening the disease. A thorough understanding of the risks and benefits of various therapeutic options is crucial in selecting the best treatment.

Treatment of melasma is very difficult and several topical hypopigmenting agents have been developed and widely used. To date, the most effective treatment is a triple-combination cream that contains 4% HQ (hydroquinone), 0.05% tretinoin and 0.01% fluocinolone acetonide. HQ is the most commonly used tyrosinase inhibitor. Tretinoin is used as an anti-wrinkle agent. However, it is also reported that topical retinoid is effective in the treatment of pigmentary disorders or can be combined with other topical agents. The third ingredient featured in triple combination products is corticosteroids. Steroids are effective in the suppression of cytokines such as endothelin-1 and granulocyte macrophage colony-stimulating factor (GM-CSF), which mediate ultraviolet (UV)-induced responses.Thus, it should be noted that triple-combination creams are effective for melasma, but can have a high frequency of adverse reactions.

Topical Hypopigmenting Agents for Pigmentary Disorders and Their Mechanisms of Action


In 1979 was discovered the ability of kojic acid to suppress tyrosinase activity. This is probably due to the ability of KA to chelate the copper component of this enzyme.
KA shows a competitive inhibitory effect on the monophenolase activity and a mixed inhibitory effect on the diphenolase activity of tyrosinase.
In a study involving 40 chinese women with epidermal melasma two sets of treatment gels were compared, one containing 2% hydroquinone and 10% glycolic acid and the other containing 2% kojic acid in the same formulation (ie., 2%hydroquinone and 10% glycolic acid).
Improvement in melasma was seen at week 4, and this continued throughout the study. At the end of the study, all patients showed improvement in their melasma, regardless of whether kojic acid was used or not.
Seventeen out of 40 (42.5%) patients had a more dramatic reduction in melasma on the side receiving kojic acid (in 2% hydroquinone and 10% glycolic acid and gel) and 5 out of 40 (12.5%) patients had a more dramatic reduction in melasma on the side receiving the hydroquinone-glycolic acid gel without kojic acid. All patients experienced redness, stinging, and mild exfoliation. Side effects were seen on both halves of the face regardless of whether kojic acid was added or not.
Kojic acid is as effective as hydroquinone in reducing the pigment in melasma. However, neither was effective in clearing the melasma completely.

Side effects experienced were acceptable by most patients, and these disappeared by the first month. Addition of kojic acid did not cause more irritation.
Both kojic acid and hydroquinone are tyrosinase inhibitors preventing the conversion of tyrosine to melanin.
The combination of both agents augment this inhibition further. Hence in patients whose melasma do not respond to either hydroquinone or glycolic acid or both, Kojic acid could be added to the treatment regime.
The addition of glycolic acid enhances penetration of both agents and hence promotes efficacy.
However, its use in cosmetics has been limited, because of the skin irritation caused by its cytotoxicity and also instability during storage.

Treatment of Melasma Using Kojic Acid in a Gel Containing Hydroquinone and Glycolic Acid


The development of novel, potent, non-toxic and stable tyrosinase inhibitors is of great importance in the medical, cosmetic and agricultural fields (18). Hence, the study was set to synthesize some derivatives of iron and copper chelating 3-hydroxypyridinone and to assess their inhibitory effect on the tyrosinase enzyme activity. It was speculated that by increasing the chelating capacity of KA its inhibitory activity will increase accordingly.

Treatment of KA (5-Hydroxy-2-(hydroxymethyl)-4H-pyran-4-one) with benzyl chloride resulted the protected form of this molecule (I) which was then reacted with ammonia or methyl amine to produce the corresponding pyridin-4(1H)-one derivatives (IIa,b). Oxidation of these alcohols with activated manganese (IV) oxide in dioxane afforded the desired aldehydes (IIIa,b). These prepared aldehydes were used as intermediates for the preparation of some 5-hydroxy pyridine-4-one derivatives containing appropriate moieties for stabilizing the radical species which would be formed from the reaction of these compounds with free radicals. Thus, IIIa and IIIb were reacted with aniline or 2-aminophenol to afford the desired final compounds after debenzylation of the 5-benzyloxy group of the obtained molecules. Tautomerization occurs in compounds IIIa, IVa and IVb.

From the experimental data it appears that all of the synthesized compounds have inhibitory effect on tyrosinase activity for the oxidation of L-DOPA, although KA is stronger.

The calculated IC50 (concentration of an inhibitor needed to inhibit half of the enzyme activity) values are reported in Table 1. It appears that the potency of inhibitory activity of compounds to be in the order of KA> V’a> Va> V’b> Vb.

Compounds Va, Vb, Va’ and Vb’ contain 2-hydroxyphenylamino or phenylamino substituent on the C-2 position of the pyridinone ring that results in a reduction in the hydrophilicity and hence a decrease in the inhibitory activity compared to the original compound. This mean that a bulky moiety attached to KA may hinder its approach to the active site of tyrosinase.
In consideration of our results, free hydroxyl group at the C-3 and C’-2 positions of the inhibitor seems to be the most important factor for the inhibition of tyrosinase activity. Similarly it has been reported that a hydroxyl group at the C-3 and C-4 positions in flavonoids is the most important factor for their tyrosinase inhibitory activity (25). Miyazawa and coworkers reported that the presence of hydroxyl groups at the C-6 and C-7 positions of coumarin skeleton plays an important role in the expression of tyrosinase inhibition (26). Also, the additional hydroxyl group in quercetin caused it to be a more potent tyrosinase inhibitor than kaempferol (27).

From these results, we conclude that hydroxyl moiety seems to be an effective functional group for the inhibition of tyrosinase.

Hence, amongst the compounds synthesized in this study, Va and V’a were more potent than Vb and V!b because they had two free hydroxyl groups in their structure compared to the latter that have only one hydroxyl group.

Furthermore, the presence of a methyl group substitute at position N1 of the hydroxypyridinone ring (ie V ‘a and V’b) caused a stronger tyrosinase inhibitory effect. The reason for this inductive effect of methyl group is probably that it pushes more electrons to the oxygen atom attached to the C-4 position of the ring making it a better ligand for the copper chelating in the active site of the enzyme.

Synthesis and tyrosinase inhibitory properties of some novel derivatives of kojic acid


Melasma is a difficult condition to treat, but it has been discovered numerous compounds able to improve patients’ conditions. Notably, Kojic Acid is as effective as hydroquinone in reducing the pigment in melasm, but neither was effective in clearing the melasma completely. Even if side effects were tolerated in the study we presented, the limitations in cosmetic use of Kojic Acid, due to its irritating effect on the skin, led the scientific research to the discover of some novel derivatives of this acid, less powerful but also less aggressive on the skin.
All these compounds are effective in melasma treatment, even if with different efficacy, and combinating them we can minimize side effects reaching a satisfying control of this medical condition.

Marco Defilippi
Gabriele Fasano

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