P. EMBLICA EXTRACT AND PRO-COLLAGEN SYNTHESIS 401 Figure 3. Effect of emblica extract on type I pro-collagen expression level. Cells were treated with various concentrations of emblica extract (0, 0.01, 0.1, 0.5, 1, and 2 mg/ml) for 24 hours, and cell lysates were sub- jected to Western blot analysis detection with specifi c antibody for type I pro-collagen protein. All Western blot results were quantifi ed using analyst/PC densitometry software. Mean densitometry data from three inde- pendent experiments were normalized to result in cells in the control. The data are represented as mean ± SD and were analyzed by the Student’s t-test. *Signifi cant difference, p 0.05, compared to untreated control. Figure 4. Effect of emblica extract and asiaticoside on type I pro-collagen expression level. Cells were treated with 0.1 mg/ml emblica extract or 0.1 mg/ml and 0.5 mg/ml asiaticoside for 24 hours, and cell lysates were subjected to Western blot analysis detection with specifi c antibody for type I pro-collagen protein. All Western blot results were quantifi ed using analyst/PC densitometry software. Mean densitometry data from three inde- pendent experiments were normalized to result in cells in the control. The data are represented as mean ± SD and were analyzed by the Student’s t-test. *Signifi cant difference, p 0.05, compared to untreated control.

JOURNAL OF COSMETIC SCIENCE 402 DISCUSSION Skin aging is a process associated with the alteration of collagen and elastin contents in dermis. Among these, type I collagen, the major component of the dermis, has been shown to be degraded in response to several environmental stimuli, including UV-radi- ation and ROS. An increase in this protein’s degradation and a decrease in its regenera- tion are considered major causes of wrinkle formation (3). Emblica fruit or Phyllanthus emblica possesses several pharmacological properties attributable to its high vitamin C content and biologically active tannins. The present study found that emblica extract dramatically increased intracellular type I pro-collagen levels in primary mouse fi bro- blasts. Moreover, emblica extract showed a strong inhibitory effect against collagenase activity. Previous studies have reported that ascorbic acid (vitamin C) was able to induce collagen synthesis (14,15). The mechanism by which vitamin C induced collagen synthesis has been shown to involve its pro-oxidant activity. This pro-oxidant effect contributes to the lipid peroxidation that is considered a primary cause of collagen induction (16). Al- though emblica fruit contain a “high level” of vitamin C, neither lipid peroxidation nor cytotoxicity was detected under the condition of pro-collagen induction in the present study. Our results suggest that the pro-collagen stimulating effect of emblica extract may not be associated with its high vitamin C content. In addition, our results show that em- blica has a greater potency than the known collagen enhancer asiaticoside. In summary, the present study indicates the novel effects of emblica extract as a pro-collagen regenera- tor as well as a collagenase inhibitor. ACKNOWLEDGMENTS The authors acknowledge a grant from the National Research Council of Thailand (NRCT) (to U. Nimmannit) and a Chulalongkorn University Research Grant for devel- Figure 5. Effect of emblica extract on collagenase activity. Quantifi cation of anti-collagenase activity of emblica extracts was determined by using an EnzChek® gelatinase/collagenase assay kit. The percent colla- genase inhibition at indicated concentrations of emblica extract obtained from three independent experi- ments is presented as mean ± SD. *Signifi cant difference, p 0.05.