Melanin, the dark pigment in the skin, is produced in the basal layer of the epidermis by specialised cells, the melanocytes, and transported, following its complete formation, to the upper layers of the epidermis where it enters into skin cells (keratinocytes) to give them their typical colour.
Ageing-associated accumulation of melanin in the upper layer of the skin is the main cause for pigmentation disorders, which is observed in Asian skin as uneven hyperpigmentation at younger ages (<30 years, often <20 years), while often seen in Western skin at a later age (50-60 years). The desire for a more youthful, more even, slightly lighter skin traditionally has been strongly held by Asian consumers. However, in more recent years, the wish for such skin has become a global phenomenon – sought are anti-ageing treatments to inhibit older age skin pigmentation or “brown spots”. Melanin, in its complete structure, is a very durable compound and there is almost no way to destroy its structure for prevention of skin colouring. The usual treatments for skin whitening today, therefore, are directed not to the decomposition of the pigment, but to the prevention of its formation, by blocking of its biosynthesis. Otherwise, random non-selectively eradication of coloured skin cells is used. These treatments suffer from obvious disadvantages:
- The needed duration of treatment, before any lightening effect can be observed, is long. It takes time for existing melanin in the upper layers of the skin to naturally fade away before the blocking of newly synthesised melanin can show its effect.
- The ingredients used for eradication of coloured cells are toxic (mercury, hydroquinone) and highly regulated or not allowed.
Unique approach
During years of research, scientists have identified the unique capacity of lignin peroxidase (LiP), a naturally occurring enzyme, to decompose melanin. The role of the LiP enzyme in this patented technology1 is to decompose and fade the melanin in its final structure after it has been already transported to the upper layer of the skin. Enzymes are biologically active compounds which can specifically target and neutralise damaging substances in the body. The important feature of enzymes is their extreme specificity for the task they were designed to perform in nature. Fundamentally, they will carry out only one specific job and will execute it in the most accurate manner, without participating in any other side reactions. The LiP dermal system, due to its unique biological enzyme power, identifies the melanin in the upper layer of the skin and specifically decomposes the pigment without affecting the biosynthesis of the melanin. The effect is, therefore, seen after a relatively short time and maintained as long as needed with repeated use. The enzyme lignin peroxidase, is a glycoprotein active at pH 2-5 and inactive above (See Fig. 1). The pH of the skin is around 5.5 with slight variations (between 5.0 and 6.5). When applied on the skin, the products containing the enzyme and the activator have to be slightly acidic and buffered for the enzyme to perform – preferably the pH will be 3.5 in order not to create skin irritation. Additionally, the enzyme requires to be first oxidised by H2O2, then reduced by a substrate molecule, e.g. veratryl alcohol, before the melanin is oxidised (Fig. 2). After application of the cream, the skin pH is temporarily reduced to 3.5 but increases afterwards to its normal level of around 5.5, thanks to the skin buffer system. At that level the enzyme is inactivated. It becomes a simple glycoprotein and, as such, will be hydrolysed in the skin, by the proteases, lipases and other glycosidases present naturally, into amino acids. Amino acids are intrinsic to the skin and the body and do not have any specific toxicity. Extensive tests have been performed to assess the safety of the LiP enzyme:
• Primary skin irritation (48 h single patch) on 50 healthy volunteers with no reaction observed.
• Primary sensitive skin irritation (48 h single patch) on 11 volunteers with sensitive skin (reacting to a 10% solution of lactic acid) with no reaction observed.
• Cumulative skin irritation was derived from the hRIPT test after 9 repeated 24 h patch applications on the same site on 50 healthy human volunteers with no reaction observed.
• Skin allergy induction: a human repeated insult patch (hRIPT) test was performed on 50 healthy volunteers and results show that there were no reactions at all during the test. As such LiP is not allergenic.
• Eye irritation: a modified Draize rabbit test was conducted according to OECD test guideline # 405 on 3 young rabbits by instillation of a LiP solution into the conjunctival sac. Results show a mild to moderate early onset and transient ocular changes such as reddening of the conjunctive and sclera discharge and chemosis. These effects were reversible and were no longer evident after 7 days. No abnormal findings of the cornea or iris were observed at any time. No corrosion observed. The test article met the classification criteria (EU Commission Directive 2001/59/EC, August 06, 2001 (Grading of Ocular Lesions)) and LiP is considered to be non-irritating to the rabbit eye.
From the above tests’ data we can conclude that the LiP enzyme formulated into a cosmetic product will not induce skin primary irritation, cumulative irritation or allergy nor eye irritation beyond what is usual for cosmetic products.
Toxicity
Dermal acute toxicity: the tests performed on LiP show the absence of dermal acute toxicity (up to 2,000 mg/kg body weight); based on the calculation the Margin of Safety (MOS) is >17,000 i.e. the ingredient is extremely safe when applied on the skin.
Panellists
Human use tests: Products containing LiP were used by healthy volunteers to assess the efficacy of the products. During all the tests, over 100 panellists have used cosmetic formulations containing LiP without one report of adverse reaction.
Efficacy
In a comparative study on Filipinos performed under the control of a dermatologist at the Souraski Medical Center in Tel-Aviv, Israel, a lotion containing LiP was applied on one arm of each of 12 volunteers. On the other arm a 2% hydroquinone cream was applied. The results showed that, after 21 days, a significantly higher lightening of the skin with LiP than with hydroquinone (Fig. 3). In another study performed in Bangkok on 22 volunteers, a lotion with LiP was applied twice daily. The Mexameter (Courage + Khazaka) results show a reduction of the melanin index after 28 days. Furthermore, the dermatologist assessment showed a significant reduction of mottled hyperpigmentation, of age spots and of sallowness. Fairness scores are increased (Fig. 4). The UV pictures taken at D0 and D28 (Fig. 5) show the improvement in skin colour and in evenness, and the decrease in age spots.
Conclusion
LiP enzyme provides a unique, innovative approach to skin lightening, eliminating the melanin in the upper layers of the epidermis. The enzyme is safe and effective. The studies showed marked improvement in the decomposition of the dark coloured pigment by LiP treatment as early as after one week of treatment and further improvement after four weeks. Elimination of visible brown spots and uneven hyperpigmentation, associated with ageing and sun damage, was observed when compared to baseline, and was significantly better when tested against competitive products.
Reference
1 USPTO Patent Application 20060051305, Methods of producing lignin peroxidase and its use in skin and hair lightening.
