]. Cosmet. Sci., 59, 431-440 (September/October 2008) Red pigment from Lithospermum erythrorhizon by supercritical CO 2 extraction HWA-YOUNG LEE, YOON-JUNG KIM, EUN-JUNG KIM, YOUNG-KEUN SONG, and SANG YO BYUN, Cosmetic R&D Center, Amore Pacific Inc., Yongin, Gyeonggi, 449-729, Republic of Korea (H.-Y.L., Y.:f.K., E.:f.K.) and Department of Molecular Science & Technology, (Y.-K.S., S. Y.B.) and Applied Biotechnology Program, Graduate School, (S. Y.B.), Ajou University, Suwon, Gyeonggi, 443-749, Republic of Korea. Accepted for publication April 9, 2008. Synopsis In this study, a stable red pigment was prepared from Lithospermum erythrorhizon via supercritical carbon dioxide extraction. The optimal extraction conditions were 400 bar and 60 ° C. The patch tests indicated that up to 10% of the red pigment was acceptable from a skin irritation standpoint. According to the results of the CIE LAB chromaticity test, the color difference was acceptable when compared to commercial synthetic red pigments. The light-illuminated color stability test indicated that the pigment was more stable than the red pigment extracted with ethanol. The higher stability was also demonstrated in the DPPH antioxidant activity test. The supercritical red pigment harbored elevated amounts of shikonin and derivatives, and appears to be usable as a stable red pigment for cosmetic color products. INTRODUCTION Lithospermum erythrorhizon Sieb. et Zucc., a traditional Chinese medicinal perennial herb, harbors shikonin and shikonin derivatives, which exert many beneficial effects, including wound-healing, antiinflammatory, antibacterial, antitumor, antidiabetes, and antiviral effects (1-3). Shikonin accumulates solely in the roots of the plant, and is one of the primary bioactive components that can be utilized in clinical settings (4,5). The prin­ cipal derivatives in the roots include acetyl shikonin, shikonin, alkanin, and other shikonin derivatives. Shikonin and alkanin are both naphthaquinone dyes with an in­ tense red color. In Japan, in 1983, the first commercial shikonin production system was initiated in a bioreactor for plant cell cultures, and certain shikonin-based perfumery/ cosmetic goods began to be produced. The chemical and consumer characteristics of shikonin generated via biotechnological methods are analogous to those in the shikonin extracted from intact plants (6,7). Address all correspondence to Sang Yo Byun. 431

432 JOURNAL OF COSMETIC SCIENCE Despite the many advantages of natural pigments for cosmetic applications, including their generally low chemotoxicity and carcinogenicity, little progress has been made with regard to the development of natural pigments. Both photo- and thermo-instability have been obstacles to the development of natural pigments for use in cosmetic appli­ cations (8,9). Another obstacle has been the high cost intrinsic to the manufacture of these pigments. This is primarily attributable to the fact that the pigments must be extracted from the plants using specific solvents, most notably alcohols. The solvent extraction of specific pigment components has generally also entailed an oxidative browning, which renders the final product inappropriate for cosmetic applications. Residual solvents remaining after extraction and evaporation also have been recognized as a factor of chemotoxicity. Supercritical carbon dioxide is now well established as a solvent that can be utilized in the extraction of raw materials for cosmetic applications. This method has a number of advantages. First, the method generally allows for quicker penetration of solid samples than is the case with liquid solvents, due to higher diffusion rates, and this method also allows for the rapid transport of dissolved solutes from the sample matrix due to low viscosity. Almost no solvent residues are present in the products. It is also, of course, conducted at low extraction temperatures, which lessens the denaturalization of the bioactive natural products (10-13). In this study, supercritical carbon dioxide was applied to the extraction of a natural red pigment from the roots of Lithospermum erythrorhizon Sieb. et Zucc. Characteristics associated with the extraction performances, yield, and shikonin and shikonin derivative contents were compared with those observed after conventional ethanol extraction. Various tests with a lip gloss prepared using the supercritical extract were conducted, in order to verify the potential of the natural red pigment for use in cosmetic applications. MATERIALS AND METHODS EXTRACTS FROM ROOTS OF LITHOSPERMUM ElffTHRORHIZON SIEB. ET ZUCC. Dried roots of Lithospermum erythrorhizon Sieb. et Zucc., which was grown in China, were ground for extraction. For supercritical carbon dioxide extraction, an apparatus includ­ ing a one-liter extractor was used, and is shown in Figure 1. Ground Lithospermt✓m erythrorhizon root powder was filled and packed into the extractor. Liquid carbon dioxide was compressed and supplied to the extractor using a pump. The temperature of the compressed supercritical fluid was adjusted with the heat exchanger prior to its intro­ duction to the extractor. Various combinations of supercritical carbon dioxide pressure and temperature were applied in an effort to optimize the extraction. After extraction, the pressure of the carbon dioxide-containing extract was reduced using the backpressure regulator. It was then separated into gaseous carbon dioxide and extracts in the separator. The liquid-phase extract was harvested from the separator. The gaseous carbon dioxide was liquefied with the chiller and recycled via supply to the compression pump. For conventional ethanol extraction, the same ground root powder was extracted for 24 hours with 95% ethanol at room temperature. The extract was lyophilized after the ethanol had been removed with a rotary vacuum evaporator.