SKIN DAMAGE PROTECTION BY PRUNUS PERSICA 31 evaluated with one-way ANOVA. P values less than 0.05 were regarded as significantly different. RESULTS AND DISCUSSION In order to develop new plant materials providing protection from UV-induced skin damage for safer and long-term use, P. persica extract (Ku-35) was evaluated in in vivo animal models that included UVB-induced erythema and edema formation. From the flowers of P. persica, several constituents such as afzelin, quercitrin, multiflorin A and B, multinoside A, and chromogenic acid were previously isolated (6). In the present investigation, four flavonol glycosides (compounds I-IV) were successfully iso- lated from the extract. Compound I was structurally confirmed based on •H-, •3C-, and COZY NMR analysis as kaempferol 3-O-[[3-D-glucopyranosyl(1--4)-ot-L- rhamnopyranoside] or multiflorin B. The •3C-NMR spectrum was well matched with the results of that previously described (7,8). Multiflorin B was initially isolated from Rosa multiflora (9). Since a glucopyranosyl(1--4)rhamnopyranosidic bond of flavonoid glycoside is very rate in plants, compound I may be used as a standard compound for the P. persica extract. Compounds II and III were structurally identified as kaempferol-3- O-[3-D-galactopyranoside (trifolin) and kaempferol-3-O-ot-L-rhamnopyranoside (afze- lin), respectively, by comparison of the spectral results of the published data (9,10). Compound IV was directly compared with an authentic standard isolated previously from the flowers of Carthamus tinctorius (11) and identified as kaempferol-3-O-[•-D- glucopyranoside (astragalin). Figure 1 demonstrates the elution profiles of the flavonoid Compound I Compound II Compound III Compound IV • 5ompou_•nd I + II + Ili + IV •_• Ku-35 Iti R•tmfion Time (rain) 20 40 6O Figure 1. HPLC elution profiles of the isolated flavonoids and P. persica extract.

32 JOURNAL OF COSMETIC SCIENCE Table II The Contents of Kaempferol Glycosides in the Extract (Ku-35) of the Flowers of P. persica Compound I II III IV Contents (%) 3.3 + 0.5 0.6 + 0.2 1.8 + 0.2 2.0 + 0.3 From 1 g of the dried flowers ofP. persica, 120 + 25 mg of dried Ku-35 were obtained (n = 3). The contents represented here are percentages of the isolated compounds based on the weight of Ku-35 (w/w, n = 3). glycosides isolated and the extract (Ku-35) in HPLC. The contents of compounds I-IV in Ku-35 are represented in Table II. Among the four kaempferol glycosides, the multiflorin B content was highest. This result also supports our suggestion that mul- ti florin B may be used as a standard compound of Ku-35. Table III demonstrates the protective effects of Ku-35 on UVB-induced erythema for- mation in guinea pig skin. In this experiment, the erythema formation gradually in- creased up to 6 hr. After this point, the intensity did not drastically change until 72 hr. Ku-35 dose dependently inhibited the erythema formation up to 72 hr. The IC5o value of Ku-35 was determined to be 0.5 mg/cm i at 6 hr after UVB irradiation by linear regression analysis, while indomethacin (0.1 mg/cm 2) showed significant inhibition of the erytherna formation at early time points. At 5.0 mg/crn 2, Ku-35 completely blocked the erythema formation (Figure 2). It is noteworthy that the inhibitory effect of Ku-35 at concentrations -- 2 mg/cm 2 was well maintained during the experiment, whereas the inhibitory activity by indomethacin gradually decreased from 24 hr after UVB irradia- tion up to 72 hr (data not shown). Multiflorin B also inhibited the erytherna formation (80%) at the dose of 0.3 mg/cm 2, suggesting that this compound is one of the active principles of Ku-35. When the inhibitory effects of Ku-35 on UVB-induced ear edema were evaluated, they also showed reduction of ear edema at doses of 0.3-3.0 mg/ear (49% inhibition at 3.0 mg/ear), as shown in Figure 3. All results obtained above clearly demonstrated for the first time, that Ku-35 protected the formations of UVB-induced erythema and edema in vivo by topical application. Previously, Ku-35 inhibited the release of arachidonic acid (AA) metabolites from UVB-induced human keratinocytes in culture (3). The inhibitory activities of Ku-35 against in vivo UVB-induced erythema and edema formation may be partly due to the inhibitory activity of the release of AA metabolites, since AA and its major metabolite, Table III Effect of P. persica Extract (Ku-35) on UVB-Induced Erythema in Guinea Pigs mg/cm 2 1 hr 4 hr 6 hr 72 hr UVB -- 1.050.0 2.0ñ0.0 3.0ñ0.0 2.5ñ0.5 Indomethacin 0.1 0.0ñ0.0' 0.5ñ0.5* 1.050.7' 2.0ñ0.5 Ku-35 0.2 0.8ñ0.4 1.3ñ0.4 2.0ñ0.7 2.5ñ0.7 1.0 0.3ñ0.4 0.5ñ0.5* 0.8ñ0.4* 1.0ñ0.7 2.0 0.0ñ0.0' 0.3ñ0.4* 0.5ñ0.5* 0.5ñ0.5* 5.0 0.0ñ0.0' 0.0ñ0.0' 0.0ñ0.0' 0.0ñ0.0' MultiflorinB 0.3 0.5ñ0.5* Numericals in this table indicate the degree of erythema: 0 = none, 1 = mild, 2 = moderate, and 3 = severe (n = 4). * P 0.05, significantly different from UVB-irradiated group by one-way ANOVA test.