Tyrosinase inhibition by extracts and constituents of Sideroxylon inerme L. stem bark, used in South Africa for skin lightening.
OBJECTIVE
To investigate the stem bark of Sideroxylon inerme L. and its compounds for tyrosinase-inhibition activity and to evaluate the mechanism involved of the most potent compounds in tyrosinase inhibition.


METHODS
Three different extracts (acetone, methanol and dichloromethane) of Sideroxylon inerme L. were evaluated for their inhibitory effect in vitro on the monophenolase and diphenolase activated forms of tyrosinase, using a colorimetric procedure. This test was used for bioactivity-guided isolation of two active compounds using column chromatography and TLC. Active extracts were also investigated for their inhibitory effect on melanogenesis in cultured B16 melanoma cells. Antioxidant activities of the methanolic extract of Sideroxylon inerme and purified compounds were investigated using the 1,2-diphenyl-2-picrylhydrazyl (DPPH) antioxidant assay. The inhibition of tyrosinase activity relative to the inhibition of its activity at the transcriptional level was also studied by determination of the degree of expression of mRNAs for this gene by using extract of Sideroxylon inerme-treated cells (B16F10) and semi-quantitative RT-PCR.


RESULTS
Methanolic and acetonic extracts of the stem bark of Sideroxylon inerme showed significant inhibition of monophenolase activity (IC50 values of 63 microg/ml and 82 microg/ml, respectively). The methanolic extract also exhibited 37% reduction of melanin content at 6.2 microg/ml in melanocytes without being significantly toxic to the cells. Examination for inhibition of monophenoloxidase in situ on TLC, followed by column chromatographic purification of the stem bark extract of Sideroxylon inerme, resulted in the isolation of two active compounds, epigallocatechin gallate and procyanidin B1, with IC50 values against monophenolase of 30 microg/ml and > 200 microg/ml, respectively. Epigallocatechin gallate exhibited a greater anti-tyrosinase activity than arbutin. Sideroxylon inerme bark extracts, epigallocatechin gallate and procyanidin B1 exhibited antioxidant DPPH radical scavenging activities with EC50 values of 1.54 microg/ml, 1.33 microg/ml and 1.68 microg/ml, respectively and were not particularly cytotoxic. During mechanism studies it was evident that at the transcription level, Sideroxylon inerme (25 microg/ml) was acting as a potent tyrosinase inhibitor compared to controls (untreated cells and kojic acid).


CONCLUSIONS
The bark extract of Sideroxylon inerme and the two isolated compounds warrant further investigation in clinical studies to be considered as skin-depigmenting agents.
Department of Plant Science, University of Pretoria, Pretoria 0002, South Africa.
S
S
Momtaz
B M
BM
Mapunya
P J
PJ
Houghton
C
C
Edgerly
A
A
Hussein
S
S
Naidoo
N
N
Lall
D000818:Animals; D000975:Antioxidants / Q000494:pharmacology; D001713:Biphenyl Compounds; D002470:Cell Survival / Q000187:drug effects; D003124:Colorimetry; D017931:DNA Primers; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D007202:Indicators and Reagents; D008543:Melanins / Q000378:metabolism; D008544:Melanocytes / Q000187:drug effects / Q000378:metabolism; D051379:Mice; D014442:Monophenol Monooxygenase / Q000037:antagonists & inhibitors*; D010851:Picrates; D024301:Plant Bark / Q000737:chemistry; D010936:Plant Extracts / Q000737:chemistry / Q000494:pharmacology; D018547:Plant Stems / Q000737:chemistry; D011753:Pyrones / Q000737:chemistry / Q000494:pharmacology; D012313:RNA / Q000096:biosynthesis / Q000302:isolation & purification; D020133:Reverse Transcriptase Polymerase Chain Reaction; D029632:Sapotaceae / Q000737:chemistry*; D012880:Skin Pigmentation / Q000187:drug effects*; D013019:South Africa
D000975:Antioxidants; D001713:Biphenyl Compounds; D017931:DNA Primers; D004791:Enzyme Inhibitors; D007202:Indicators and Reagents; D008543:Melanins; D010851:Picrates; D010936:Plant Extracts; D011753:Pyrones; D012313:RNA; C011890:kojic acid; C004931:1,1-diphenyl-2-picrylhydrazyl; D014442:Monophenol Monooxygenase