554 JOURNAL OF COSMETIC SCIENCE constantly until an emulsion formed. The 2% hydroquinone solution in propylene glycol was added to the cream at 40øC, and the resulting mixture was stirred while cooling to room temperature. The incorporation of licorice extract or commercial antioxidants to the formulation during preparation depends on the solubility properties. The extract (0.1%, 0.5%, 1.0%, and 2.0% w/w), levigated by propylene glycol (5% w/w) and BHT (0. ! %, 0.5 %, 1.0%, and 2.0% w/w), was in the oil phase, whereas sodium metabisulfite (0.t%, 0.5%, 1.0%, and 2.0% w/w) was in the water phase. The control was 2% hydroquinone cream without the extract or any commercial antioxidants. ANTIOXIDATIVE ACTIVITY STUDY A 10-g sample was put into a 20-ml, tightly screw-capped, test tube. One set of test samples was incubated at 45 ø + 0.5øC in an incubator (Fanazma Incubator, Iran) for three months to evaluate formulation stability. Another set was kept in a dark room at 25 ø + 0.5øC for three months. Samples at each concentration of the extract and com- mercial antioxidants were done in triplicate. Physical stability behaviors, i.e., changes in color and separation of emulsion, were observed optically every week. For the determi- nation of the average percentages of hydroquinone remaining at 25 o + 0.5 øC and at 45 o + 0.5øC after two weeks and after one, two, and three months, one gram of the tested samples was extracted with methanol and the amount of hydroquinone was measured by a UV spectrophotometer (Spectronic Genesys 2, USA) at 294 nm, according to the official standard hydroquinone assay (1,19). A B 100 100 90 90 80 80 30 30 20 20 10 10 0 0 , . ..... 0 2 4 6 8 10 12 0 2 4 6 8 10 12 Time (Week) Time (Week) ß CB •Ext. 0.1% •. CB •Ext. 0.1% & SM 0.1% ',4: BHT0.1% & SM 0.1% ,( BHT0.1% Figure 2. Formulation stability study of 2% w/w hydroquinone cream containing 0.1% extract and commercial antioxidants incubated at 25 ø + 0.5øC (A) and 45 ø _+ 0.5øC (B) for three months.

ANTIOXIDANT ACTIVITY OF LICORICE EXTRACT 555 STATISTICAL ANALYSIS ANOVA testing followed a Tukey test used to determine significant differences between groups, and p ( 0.05 was considered significant. RESULTS AND DISCUSSION Powdered dry roots gave a yield of 21.0% w/w of dried licorice extract. The licorice extract is yellowish-brown in color. From physical observation (data not shown), the color of all sample systems was darker starting from the first week, with the darkest intensity in cream without any antioxidants (CB+HY) and the least dark in the system with 2.0% extract. The systems containing 2.0% sodium metabisulfite (SM) and !.0% and 2.0% BHT showed emulsion instability. The 2.0% BHT cream started to crack from the first month, whereas 2.0% SM cream started to crack at the second month and continued. This may be due to the high concentrations of both antioxidants compared with the other test systems. BHT is a phenolic antioxidant for fatty acid and vegetable oil. Usually it is used at a level of 0.01-0.1% in cosmetics containing unsaturated materials (1). The extract systems at all concentrations (0.1%, 0.5%, 1.0%, and 2.0% w/w) were stable for three months. When comparing the percentages of hydroquinone remaining after incubation at dif- ferent temperatures for three months, the sodium metabisulfite, BHT, and licorice extract systems in all concentrations in the dark room at 25øC showed a higher remain- A 100 100 90 90 80 80 •o .• •o .o .o !.o 20 [ 20 10 10 0 ...... 0 0 2 4 6 8 10 12 B 0 2 4 6 8 10 12 Time (Week) Time (Week) •CB 0.5% [ C CB 0.5% L '-- SM 0.5% ..( •Ext. BHT o.5%j •. SM 0.5% ..( •Ext. BHT 0.5% Figure 3. Formulation stability study of 2% w/w hydroquinone cream containing 0.5% extract and commercial antioxidants incubated at 25 ø _+ 0.5øC (A) and 45 ø _+ 0.5øC (B) for three months.