中醫藥是中華民族的瑰寶，近年來傳統中醫藥被西方國家消費者之接受度逐漸提升，主要原因與全球掀起回歸自然的熱潮有關，且中藥方劑是組合療法(combined therapy)，與合成藥物相比，產生較少之副作用。研究證實中藥複方療效往往明顯優於單味藥物與單一成分，因此不能單純地認為中藥方劑中主要藥味的主要成分即為複方的有效成分，亦無法應用西藥的尺度來衡量中藥單體成分的活性。紫雲膏為一主要用於外傷(割傷、擦傷、切傷)、燙傷、特異性皮膚炎之外科漢方，主要由紫草、當歸、黃蠟、胡麻油所組成。紫草已被證實具有加速肉芽組織生長、促進傷口癒合、抗菌之作用，而當歸則具有提高機體免疫和造血功能，促進蛋白質和核酸代謝，改善血液流變性等作用。
在評估外用皮膚製劑生體相等性方面，自1990年代以來，美國藥物食品檢驗局即建議並嘗試開發以皮膚藥動藥效特性來做為除了臨床試驗外，得以驗証外用皮膚製劑生體相等性的另一種試驗方法。皮膚藥動試驗法（Dermatopharmatokinetics / DPK）利用皮膚層撕法和藥物活性成分之分析，可評估角質層上活性成分滲透濃度之動態變化。本研究之目的為利用皮膚層撕法探討紫雲膏各指標成分在人類角質層上的藥動變化；及利用細胞移行試驗評估測定角質層上的藥品分佈對於促進傷口癒合的能力，嘗試評估角質層上活性成分濃度與傷口相對癒合能力之關連性，以探討皮膚藥動藥效試驗法做為外用中草藥多成分製劑生體相等性試驗法之可行性。
根據角質層中紫雲膏指標成分濃度的經時變化（DPK profile），比較兩種不同廠牌紫雲膏各指標成分之各項皮膚藥動參數（Cmax、Tmax、AUC0-12、ke、T1/2）皆無統計上顯著差異。且細胞移行試驗結果亦顯示兩種不同廠牌的紫雲膏之角質層滲透濃度對WS-1細胞之相對最大癒合能力（Emax）無統計上顯著差異。進一步比較各指標成分之Cmax、AUC0-12的90％信賴區間，判斷兩種不同廠牌紫雲膏各指標成分具有生體相等性，顯示皮膚藥動試驗法應可作為外用紫雲膏製劑生體相等性評估之方法。
以複迴歸模式分析角質層上紫雲膏指標成分滲透濃度與WS-1細胞在傷口區域移行能力之關聯性，顯示紫草素Shikonin及其衍生物Dimethylacrylshikonin、α-methyl-n-butyrylshikonin與傷口區域相對癒合能力有正相關；Ferulic acid、Acetylshikonin與Isobutylshikonin則為負相關。研究結果顯示複迴歸模式可適當地描述角質層紫雲膏指標成分滲透濃度與WS-1細胞在傷口區域移行能力之關聯性，並可預測角質層上紫雲膏各指標成分對於傷口區域相對癒合能力之相對重要性。

Herbal medicines have been used in medical practices for thousands of years and recognized as a valuable resource for healthcare. In the last several decades, there has been increasing acceptance of Traditional Chinese Medicine（TCM）by consumers in the Western countries. The major reason is herbal treatments, especially in combination, produce fewer side-effects than many synthetic medications. TCM prescriptions usually consist of mixture herbs and offer a very complex combination of bioactive compounds. The complexity of the formula and the incertainty of the active ingredients are the major barrier for the standardization of herbal medicines. Shiunko, a formula originated from the Ming Dynasty of the ancient China, is composed of the roots of Lithospermi radix and Angelia sinensis. The formula was designated for the treatment of wounded skin caused by cuts, abrasions, frost, or burn. Previous studies demonstrated that Shiunko exerted an enhancing effect on the proliferation of fibroblasts, and the production of collagen fiber in the wounded skin.
Since 1990s, FDA has been proposing and exploring the possibility that a dermatopharmacokinetic and pharmacodynamic（DPK/PD）characterization might provide an alternative approach to clinical trials for the determination of bioequivalence of topical dermatologic products. DPK studies use skin stripping technique and analytical methods to assess drug concentration in the stratum corneum (SC) with respect to time. The present studies aimed to evaluate the feasibility of DPK/PD approach as a bioequivalence assessment method for topical herbal products in Chinese population. Specifically, these studies were designed to apply the skin stripping technique to determine the kinetic profiles of multiple ingredients of two Shiunko formulations in the SC and to assess the effect of drug concentration in the SC on wound-healing by cell migration assay. An effort was also made to correlate kinetic parameters with the resulting wound-healing effect.
The DPK profile of Shiunko formulation demonstrated that the DPK parameters（Cmax、Tmax、AUC0-12、ke、T1/2）of two Shiunko formulations were not statistically different from each other. Similarly, the relative maximum healed area of drug content in the tape-stripped SC to WS-1 cells determined by cell migration assay were not significantly different between the two formulations. Furthermore, the 90％ confidence interval of Cmax and AUC0-12 of six markers demonstrated the two Shiunko formulations are bioequivalent. As a result, the DPK methodology should be an appropriate method for bioequivalence assessment of Shiunko formulation.
Finally, by multiple regression, the concentrations of shikonin, dimethylacrylshikonin and α-methyl-n-butyrylshikonin were positively correlated with the resulting wound-healing effect, while the concentrations of ferulic acid, acetylshikonin and isobutylshikonin showed negative correlation. The results suggest that the model can be useful in delineating the relative contribution of individual markers in Shiunko formulation with regard to its resulting wound-healing effect.

Akimoto K, Kitagawa Y, Akamatsu T, Hirose N, Sugano M, Shimizu S and Yamada H (1993) Protective effects of sesamin against liver damage caused by alcohol or carbon tetrachloride in rodents. Ann Nutr Metab 37:218-224.
Bent S and Ko R (2004) Commonly used herbal medicines in the United States: a review. Am J Med 116:478-485.
Bonina F, Puglia C, Ventura D, Aquino R, Tortora S, Sacchi A, Saija A, Tomaino A, Pellegrino ML and de Caprariis P (2002) In vitro antioxidant and in vivo photoprotective effects of a lyophilized extract of Capparis spinosa L buds. J Cosmet Sci 53:321-335.
Chavali SR, Utsunomiya T and Forse RA (2001) Increased survival after cecal ligation and puncture in mice consuming diets enriched with sesame seed oil. Crit Care Med 29:140-143.
Chen X, Yang L, Oppenheim JJ and Howard MZ (2002) Cellular pharmacology studies of shikonin derivatives. Phytother Res 16:199-209.
Escobar-Chavez JJ, Merino-Sanjuan V, Lopez-Cervantes M, Urban-Morlan Z, Pinon-Segundo E, Quintanar-Guerrero D and Ganem-Quintanar A (2008) The tape-stripping technique as a method for drug quantification in skin. J Pharm Pharm Sci 11:104-130.
FDA (1995) Guidance: Topical dermatological corticosteroids: In vivo bioequivalence. CDER.
FDA (1998) Guidance (Draft): Topical dermatological drug product NDA's and ANDA's- In vivo bioavailability, bioequivalence and In vitro release. CDER.
FDA (2004) Guidance for Industry: Botanical Drug Products CDER.
Fluhr JW, Vienne MP, Lauze C, Dupuy P, Gehring W and Gloor M (1999) Tolerance profile of retinol, retinaldehyde and retinoic acid under maximized and long-term clinical conditions. Dermatology 199 Suppl 1:57-60.
Graf E (1992) Antioxidant potential of ferulic acid. Free Radic Biol Med 13:435-448.
Guzzo C, Lazarus G and Werth V (1996) Dermatological pharmacology. Goodman and Gilman's, New York.
Hayashi M (1977a) [Pharmacological studies of Shikon and Tooki. (2) Pharmacological effects of the pigment components, Shikonin and acetylshikonin]. Nippon Yakurigaku Zasshi 73:193-203.
Hayashi M (1977b) [Pharmacological studies of Shikon and Tooki. (3) Effect of topical application of the ether extracts and Shiunko on inflammatory reactions]. Nippon Yakurigaku Zasshi 73:205-214.
Hirose N, Inoue T, Nishihara K, Sugano M, Akimoto K, Shimizu S and Yamada H (1991) Inhibition of cholesterol absorption and synthesis in rats by sesamin. J Lipid Res 32:629-638.
Hon CC, Chow YC, Zeng FY and Leung FC (2003) Genetic authentication of ginseng and other traditional Chinese medicine. Acta Pharmacol Sin 24:841-846.
Hu HJ and Hang BQ (1991) [Effects of ferulic acid on allergic reaction]. Zhongguo Yao Li Xue Bao 12:426-430.
Hu Y, Jiang Z, Leung KS and Zhao Z (2006) Simultaneous determination of naphthoquinone derivatives in Boraginaceous herbs by high-performance liquid chromatography. Anal Chim Acta 577:26-31.
Huang KF, Hsu YC, Lin CN, Tzeng JI, Chen YW and Wang JJ (2004) Shiunko promotes epithelization of wounded skin. Am J Chin Med 32:389-396.
Li WF, Jiang JG and Chen J (2008) Chinese medicine and its modernization demands. Arch Med Res 39:246-251.
Loyd V. Allen, Nicholas G. Popovich and Ansel. HC (2005) Ansel's pharmaceutical dosage forms and drug delivery systems. Lippincott Williams & Wilkins, Philadelphia.
Lu PJ, Yang C, Lin CN, Li CF, Chu CC, Wang JJ and Chen JY (2008) Shiunko and acetylshikonin promote reepithelialization, angiogenesis, and granulation tissue formation in wounded skin. Am J Chin Med 36:115-123.
Madhujith T and Shahidi F (2007) Antioxidative and antiproliferative properties of selected barley (Hordeum vulgarae L.) cultivars and their potential for inhibition of low-density lipoprotein (LDL) cholesterol oxidation. J Agric Food Chem 55:5018-5024.
N'Dri-Stempfer B, Navidi WC, Guy RH and Bunge AL (2008) Optimizing metrics for the assessment of bioequivalence between topical drug products. Pharm Res 25:1621-1630.
Namiki M (2007) Nutraceutical functions of sesame: a review. Crit Rev Food Sci Nutr 47:651-673.
Navidi W, Hutchinson A, N'Dri-Stempfer B and Bunge A (2008) Determining bioequivalence of topical dermatological drug products by tape-stripping. J Pharmacokinet Pharmacodyn.
Ozaki Y (1992) Antiinflammatory effect of tetramethylpyrazine and ferulic acid. Chem Pharm Bull (Tokyo) 40:954-956.
Ozaki Y, Ohno A, Abe K, Saito Y and Satake M (1993) Comparative study on the accelerative effect of "koushikon" and "nanshikon" and their constituents on proliferation of granuloma tissue in rats. Biol Pharm Bull 16:683-685.
Ozaki Y, Ohno A, Saito Y and Satake M (1994) Accelerative effect of shikonin, alkannin and acetylshikonin on the proliferation of granulation tissue in rats. Biol Pharm Bull 17:1075-1077.
Ozaki Y, Sakaguchi I, Tujimura M, Ikeda N, Nakayama M, Kato Y, Suzuki H and Satake M (1998) Study of the accelerating effect of shikonin and alkannin on the proliferation of granulation tissue in rats. Biol Pharm Bull 21:366-370.
Parihar VK, Prabhakar KR, Veerapur VP, Kumar MS, Reddy YR, Joshi R, Unnikrishnan MK and Rao CM (2006) Effect of sesamol on radiation-induced cytotoxicity in Swiss albino mice. Mutat Res 611:9-16.
Pershing LK, Bakhtian S, Poncelet CE, Corlett JL and Shah VP (2002a) Comparison of skin stripping, in vitro release, and skin blanching response methods to measure dose response and similarity of triamcinolone acetonide cream strengths from two manufactured sources. J Pharm Sci 91:1312-1323.
Pershing LK, Corlett J and Jorgensen C (1994) In vivo pharmacokinetics and pharmacodynamics of topical ketoconazole and miconazole in human stratum corneum. Antimicrob Agents Chemother 38:90-95.
Pershing LK, Corlett JL and Nelson JL (2002b) Comparison of dermatopharmacokinetic vs. clinicial efficacy methods for bioequivalence assessment of miconazole nitrate vaginal cream, 2% in humans. Pharm Res 19:270-277.
Pershing LK, Nelson JL, Corlett JL, Shrivastava SP, Hare DB and Shah VP (2003) Assessment of dermatopharmacokinetic approach in the bioequivalence determination of topical tretinoin gel products. J Am Acad Dermatol 48:740-751.
Pershing LK, Silver BS, Krueger GG, Shah VP and Skelley JP (1992) Feasibility of measuring the bioavailability of topical betamethasone dipropionate in commercial formulations using drug content in skin and a skin blanching bioassay. Pharm Res 9:45-51.
Piacquadio D and Kligman A (1998) The critical role of the vehicle to therapeutic efficacy and patient compliance. J Am Acad Dermatol 39:S67-73.
Rodriguez LG, Wu X and Guan JL (2005) Wound-healing assay. Methods Mol Biol 294:23-29.
Rougier A, Dupuis D, Lotte C, Roguet R and Schaefer H (1983) In vivo correlation between stratum corneum reservoir function and percutaneous absorption. J Invest Dermatol 81:275-278.
Sakaguchi I, Tsujimura M, Ikeda N, Minamino M, Kato Y, Watabe K, Yano I and Kaneda K (2001) Granulomatous tissue formation of shikon and shikonin by air pouch method. Biol Pharm Bull 24:650-655.
Shah VP (2001) Progress in methodologies for evaluating bioequivalence of topical formulations. Am J Clin Dermatol 2:275-280.
Shah VP, Elkins J and Skelly JP (1992) Relationship between in vivo skin blanching and in vitro release rate for betamethasone valerate creams. J Pharm Sci 81:104-106.
Shah VP, Flynn GL, Yacobi A, Maibach HI, Bon C, Fleischer NM, Franz TJ, Kaplan SA, Kawamoto J, Lesko LJ, Marty JP, Pershing LK, Schaefer H, Sequeira JA, Shrivastava SP, Wilkin J and Williams RL (1998) Bioequivalence of topical dermatological dosage forms--methods of evaluation of bioequivalence. Pharm Res 15:167-171.
Shah VP, Midha KK, Dighe S, McGilveray IJ, Skelly JP, Yacobi A, Layloff T, Viswanathan CT, Cook CE, McDowall RD and et al. (1991) Analytical methods validation: bioavailability, bioequivalence and pharmacokinetic studies. Conference report. Eur J Drug Metab Pharmacokinet 16:249-255.
Shimizu S, Akimoto K, Shinmen Y, Kawashima H, Sugano M and Yamada H (1991) Sesamin is a potent and specific inhibitor of delta 5 desaturase in polyunsaturated fatty acid biosynthesis. Lipids 26:512-516.
Sidhu GS, Singh AK, Banaudha KK, Gaddipati JP, Patnaik GK and Maheshwari RK (1999) Arnebin-1 accelerates normal and hydrocortisone-induced impaired wound healing. J Invest Dermatol 113:773-781.
Siow YL, Gong Y, Au-Yeung KK, Woo CW, Choy PC and O K (2005) Emerging issues in traditional Chinese medicine. Can J Physiol Pharmacol 83:321-334.
Sitruk-Ware R (1989) Transdermal delivery of steroids. Contraception 39:1-20.
Sun GX, Hou ZF, Bi YM, Bi KS and Sun YQ (2006) [The digital criterion of the potential information characteristics of the traditional Chinese medicine chromatographic fingerprints]. Yao Xue Xue Bao 41:857-862.
Surber C, Wilhelm KP, Hori M, Maibach HI and Guy RH (1990) Optimization of topical therapy: partitioning of drugs into stratum corneum. Pharm Res 7:1320-1324.
Treffel P and Gabard B (1993) Feasibility of measuring the bioavailability of topical ibuprofen in commercial formulations using drug content in epidermis and a methyl nicotinate skin inflammation assay. Skin Pharmacol 6:268-275.
van Rooijen GJ and Moloney MM (1995) Plant seed oil-bodies as carriers for foreign proteins. Biotechnology (N Y) 13:72-77.
Wang B, Ouyang J, Liu Y, Yang J, Wei L, Li K and Yang H (2004) Sodium ferulate inhibits atherosclerogenesis in hyperlipidemia rabbits. J Cardiovasc Pharmacol 43:549-554.
Weigmann H, Lademann J, v Pelchrzim R, Sterry W, Hagemeister T, Molzahn R, Schaefer M, Lindscheid M, Schaefer H and Shah VP (1999) Bioavailability of clobetasol propionate-quantification of drug concentrations in the stratum corneum by dermatopharmacokinetics using tape stripping. Skin Pharmacol Appl Skin Physiol 12:46-53.
Wiedersberg S, Leopold CS and Guy RH (2008) Bioavailability and bioequivalence of topical glucocorticoids. Eur J Pharm Biopharm 68:453-466.
Yasuda T, Takasawa A, Nakazawa T, Ueda J and Ohsawa K (2003) Inhibitory effects of urinary metabolites on platelet aggregation after orally administering Shimotsu-To, a traditional Chinese medicine, to rats. J Pharm Pharmacol 55:239-244.
Zhang JZ and Yan Y (2004) [Application of adsorption resin separation technique in traditional China medicine research]. Zhongguo Zhong Yao Za Zhi 29:628-630.
王光清 (2001) 中國膏藥學. 陝西科學技術出版社, 陝西.
林景彬 (2001) 常用中藥藥理與應用. 中國醫藥學院, 台中.
許濘雅 (2004) Hydrocortisone 乳軟膏之皮膚藥動學研究. 國立成功大學, 台南.
陳恕誼 (2005) 利用體外釋出、皮膚層撕法及皮膚藥物動力學比較外用Tretinoin製劑. 國立成功大學, 台南.
滕佳林 and 米杰 (2004) 外治中藥的研究與應用. 上海科學技術出版社, 上海.
蔡宜芳 (2001) 外用0.05% Clobetasol 17-Propionate之藥動藥效生體相等性研究. 國立成功大學, 台南.
衛生署中華藥典編修委員會 (2000) 中華藥典第五版附錄. 行政院衛生署, 台北市.
羅佳波 (2002) 中藥傳統技術的現代研究與保護. 世界科學技術---中藥現代化 4:13-16.