BREADFRUIT EXTRACT AS SKIN LIGHTENER 49 Table III Percentage of Free-Radical Scavenging of A. incisus Ether Extract Concentration of A. incisus ether extract (µg/ml) 0.5 1 5 10 25 50 100 250 500 2000 Free radical scavenging (%) 3.64 ± 0.29 7.70 ± 0.30 10.58 ± 0.43 13.09 ± 0.33 15.35 ± 0.22 20.30 ± 1.27 41.67 ± 1.81 62.71 ± 1.34 77.89 ± 1.23 94.08 ± 0.92 Table IV EC50 (µg/ml) 169.53 ± 9.73 Percentage of Free-Radical Scavenging of 1-Ascorbic Acid and Butylated Hydroxytoluene Concentration (µg/ml) 0.25 0.5 2.5 5 25 50 125 250 1000 2500 EC50 (µg/ml) 1-ascorbic acid 7.83 ± 1.72 15.81 ± 0.53 41.02 ± 3.97 69.03 ± 0.16 88.10 ± 0.33 93.89 ± 0.12 94.01 ± 0.13 94.09 ± 0.15 93.94 ± 0.13 93.96 ± 0.38 3.16 ± 0.26 Free radical scavenging (%) Butylated hydroxytoluene 12.68 ± 0.76 15.17 ± 1.64 56.68 ± 0.91 80.51 ± 0.73 92.41 ± 1.41 94.88 ± 0.23 95.00 ± 0.08 97.02 ± 1.35 96.12 ± 0.11 94.93 ± 1.46 2.30 ± 0.03 DPPH test only recognizes free-radical scavenging effects and not pro-oxidant activity (8). Therefore, other antioxidant properties, such as reducing o-quinones or other inter­ mediates in melanin biosynthesis of the ether extract, should be taken into consideration and further clarified in the future. MELANOGENESIS-INHIBITORY ACTIVITY In this study, the activity of melanocyte B16Fl cells was investigated by determination of the melanin content of cells treated with A. incisus ether extract, purified artocarpin, or lightening agents including kojic acid and hydroquinone. The A. incisus extract showed the dose-dependent inhibition of melanin production of B16Fl cells, as shown in Figure 5. The percentages of melanin reduction in the extract-treated cells compared to that of the control (DMSO-treated) cells at concentrations of 2, 10, 15, and 25 µg/ml of A. incisus extract were 5.34%, 8.76%, 16.73%, and 26.65%, respectively. The final concentration of DMSO in the cultured medium was not more than 0.1 % v/v. To provide more evidence that the extract of A. incisus potentially inhibits melanogen­ esis, we determined its effects on melanin production in comparison to well-known

50 "'ii u CJ =a·= tu CD ._ C fl) fl) � � LL. u (I) '?ft. JOURNAL OF COSMETIC SCIENCE 100 -----· .., ... ,' , , I , . , , 50 , , , . I , I , , I � __________.,,,.- _.,,, 0 -+-----...----r------r-------.------, -1 0 1 2 3 Log concentration (µg/ml) 4 - -BHl -- L-ascorbic acid • • • • • A. incisus extract Figure 4. Antioxidant activity of A. incisus ether extract, L-ascorbic acid, and butylated hydroxytoluene. 120 Gi * (J 100 C a, 80 C (J 60 C ·2 40 Gi :l! 20 � 0 0 Control 2 ug/ml 10 ug/mL 15 ug/ml 25 ug/ml Concentration (ug/ml) Figure 5. Inhibitory effects of A. incisus ether extract on melanin synthesis in melanocyte B16Fl melanoma cells. Data are expressed as percent of control, and each column represents mean ± S.D. of triplicate study. Significantly different from the control value: *p 0.5, **p 0.01. skin-lightening agents, kojic acid and hydroquinone. The concentration of each tested sample used was 10 µg/ml, which corresponds to the IC 50 of the ether extract, according to a previous study of tyrosinase-inhibitory activity. Additionally, purified artocarpin, the major component of the A. incisus's heartwood extract, was investigated for its melanogenesis-inhibitory effect. Its concentration used was 4.5 µg/rnl, which is equiva­ lent to 4.5 µg/ml of artocarpin contained in the extract, according to a previous study of artocarpin content in the extract. The effect of the tested samples on the melanin production of melanocyte B16Fl cells is shown in Figure 6. The reduction of melanin synthesis was expressed by kojic acid, A. incisus ether extract, artocarpin, and hydroquinone as 9.61 %, 8.76%, 8.88%, and 40.97%, respectively, compared to the control cells. These results reveal that, at a similar artocarpin concentration, the inhibition of melanin production of the ether extract shows no difference from that of the artocarpin. Compared to kojic acid, it is likely that the potential of melanin reduction of the ether extract is close to that of kojic acid.