58 SKIN BRIGHTENING
colorimetric method based on the reduction of a ferric TriPyridylTriaZine (TPTZ) complex to its ferrous form. It can give indications about the capability of a substance to protect against oxidant injury when compared to a well-known antioxidant compound.12
At low pH,
reduction of ferric tripyridyl triazine (Fe III TPTZ) complex to ferrous form can be monitored by measuring the change in absorption at 593 nm. The reaction is nonspecific, in that any half reaction that has lower redox potential, under reaction conditions, than that of ferric ferrous half reaction, will drive the ferrous (Fe III to Fe II) ion formation. The change in absorbance is therefore, directly related to the combined or ‘total’ reducing power of the electron donating antioxidants present in the reaction mixture. Larix sibirica and ascorbic acid (used as
control) were tested at two concentrations in water, using the FRAP assay. Data are reported as values of reducing power of ascorbic acid fixed to 100. When compared to ascorbic acid, Larix
sibirica used at the same concentration exert very good antioxidant properties, being the latter value 70% of the value shown by pure ascorbic acid. It can be noticed that the values resemble those obtained with the 0.1% solutions indicating that the extract may exert antioxidant capacities even at extreme low concentrations (Fig 3). The results indicate that Larix sibirica is
of relevant interest as an antioxidant for lightening applications.
Lightening activity
The lightening capability of Larix sibirica (pure substance) was evaluated at two concentrations in B16 melanocyte cell culture. Kojic acid and SAP (Sodium Ascorbyl Phosphate) were used as positive control and untreated cells as negative control. The cytotoxicity level of the tested products has been determined in human dermal fibroblast (HDF) culture cells using MTT assay. Results (mean ± standard deviation) are shown in Figure 4 and 5. The cytotoxicity test indicated that Larix
sibirica does not show any toxic effect whereas Kojic acid and SAP showed slightly toxic effect at the tested concentrations on HDF cells (Fig 4). Larix sibirica inhibited melanin
production up to 67% when compared to untreated cells. Inhibitory activity of the extract on melanin production had dose- dependent response (Fig 5).
Transcriptomic analysis
The mechanism of action on inhibition of melanin biosynthesis has been described on the melanocyte specific genes Tyr and Mift, and genes involved in melanosome
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120.00 100.00 80.00 60.00 40.00 20.00 0.00
Control
Kojic Ac
Kojic Ac. 1%
SAP 1%
SAP 2%
Larix Sibirica 0.025%
Figure 4: Evaluation of Larix sibirica effect on HDF viability (cytotoxicity test).
120.00 100.00 80.00 60.00 40.00 20.00 0.00
Control
Kojic Ac
Kojic Ac. 1%
SAP 1%
SAP 2%
Larix Sibirica 0.025%
Larix Sibirica
Figure 5: Evaluation of Larix sibirica effect on melanin concentration in melanocytes. B16 melanocytes were treated for a period of 36 hours. Concentration of melanin was determined by spectrophotometry at (l) 470nm.
structure and biogenesis Pmel17 and Mart1.13
Siberian Larch extract suppressed the
expression of Tyr, Mitf, Pmel17 and Mart. Mitf, a helix loop helix–leucine zipper (HLH– LZ) transcription factor, regulates transcription of key pigmentation genes, tyrosinase (Tyr), tyrosinase related protein-1 (Tyrp1), and tyrosinase related protein-2
n Pmel17 n Mart1 n Tyr n Mitf
140 120 100 80 60 40 20 0
C SL 50 SL 25
Figure 6: Effect of Siberian larch extract (SL) on specific pigmentation genes. Effect of Siberian Larch (SL) at 25 μg/mL or 50 μg/mL on Tyr, Mitf, Pmel17 and Mart1 mRNA expression levels as determined by real time RT-PCR. Untreated Melan-a cells were used as controls (C). Results represent the mean ± SD with n=3.
(Tyrp2)/Dct (dopachrome tautomerase), and is recognised as the master regulator of pigmentation genes. Pmel17 and Mart1, key melanosome structural component genes, are critical for trafficking, appropriate folding, polymerisation, and synthesis of early stage melanin in the melanosome. The results indicate that Siberian larch
reduces melanin content, at least in part, through the suppression of key pigmentation genes, Mitf and Tyr, and melanosome structural component genes, Pmel17 and Mart1. Altogether, the results support the lightening effect of Siberian larch extract by inhibition of melanin biosynthesis and melanosome biogenesis and structure.
DNA protection by Larix sibirica The comet assay, or single cell gel electrophoresis assay (SCGE), is a common technique for measurement of DNA damage in individual cells. Under an electrophoretic field, damaged cellular DNA is separated from intact DNA, yielding a classic ‘comet tail’ shape under the microscope. Human Dermal Fibroblasts (HDF) and Human Epidermal Keratinocytes (HEK) were used to evaluated DNA protection of the Siberian Larch extract versus untreated cells, as negative control and resveratrol (15µM) as positive control.
June 2019 Larix Sibirica
% Fold change in gene expression % Melanin content % Viability
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