In Search of an Innovative Agent for Skin Care - Putting an Ancient Herbal Cosmetic Formula on Modern Bioactivity Testing Platforms

Backgroud: Qi Bai San (QBS) is a traditional Chinese herbal formula used by ancient ladies for healthy skin and whitening. Nevertheless, it contains undesirable animal and toxic herbs, without scientific evidence demonstrating its efficacy. Objective: This study aims to compare and identify QBS formula with the best efficacy from three different versions of QBS formulations, F1, F2, and F3. Methods: Cellular melanogenesis and tyrosinase activity assays were used to assess melanin content and tyrosinase activity on α-melanocytestimulating hormone (MSH)induced B16 cells. Collagenase inhibition assay was used to compare the collagenase inhibitory activity. Effects of QBS on melanin production was determined using UVirradiated Balb/c mice. Transdermal experiment was used to confirm whether QBS could penetrate into the skin. In vitro skin toxicity test study was performed to determine whether QBS would cause toxicity to skin cells. 1.4. Results: F1, F2 and F3 dose-dependently reduced α-MSH-induced increase in melanin content and tyrosinase activity, and inhibited collagenase activity. F3 is the simplest formula among all formulations (without animal or toxic herbs), yet demonstrating similar efficacy. Animal study suggested F3 could reduce melanocytes and melanin content in UVirradiated mice. Further penetration and skin toxicity studies suggested markers from different herbs within F3 could penetrate through the epidermis to exhibit its effects, without causing toxicity to skin cells. 1.5. Conclusion: We showed for the first time that a modified QBS formula exert hypopigmentation and collagenase inhibitory effects, providing in vitro and in vivo scientific evidence supporting its efficacy on hypopigmentation and healthy skin promotion. Avens Publishing Group Inviting Innovations Citation: Wat E, Siu WS, Chan HY, Ka Tso TH, Law HW, et al. In Search of an Innovative Agent for Skin Care Putting an Ancient Herbal Cosmetic Formula on Modern Bioactivity Testing Platforms. J Clin Investigat Dermatol. 2019;7(1): 5 J Clin Investigat Dermatol 7(1): 5 (2019) Page 02 ISSN: 2373-1044 Paeoniae Alba Radix (dried processed root of Paeonia lactiflora Pall.), Bombyx Batryticatus dried body of Bombyx mori Linnaeus, or the forth to fifth infected instar larvae (or by artificial inoculation) by Beauveria bassiana (Bals.) to death, Angelicae Dahuricae Radix (dried root of Angelica dahurica (Fisch. ex Hoffm.) Benth. Et Hook.), Typhonii Rhizoma, Poria (dried sclerotium of Poria cocos (Schw.) Wolf), and Tribuli Fructus (dried ripe fruit of Tribulus terrestris L.) [8]. Recent scientific studies also supported the notion that the different herbs within QBS are potent on reducing the skin colour of healthy volunteers receiving recreational exposure to sunlight [9]. Laboratory experiments demonstrated that these herbs from QBS could significantly inhibit tyrosinase activity, thereby contributing to their effects in the control of pigmentations [7]. With the several versions of the formula carrying slightly different components, and keeping an open mind on the possible compositions, we chose three formulae of slightly different combinations to be compared in our in vitro platform studies to identify the formula with the best cosmetic effects. The most efficacious formula was chosen and tested in our ex vivo and in vivo platforms to compare its effect with kojic acid alone. The three formulae were: Formula 1, consisting of Poria; Atractylodis Macrocephalae Rhizoma; Angelicae Dahuricae Radix; Paeoniae Alba Radix; Ampelopsis Radix; Bombyx Batryticatus; and Tribuli Fructus; Formula 2, consisting of the same herbs except Bletillae Rhizoma was used to replace Angelicae Dahuricae Radix; and Formula 3, consisting of only 5 herbs of Formula 1 when Tribuli Fructus and Bombyx Batryticatus were removed. Overall planning of investigation To explore the cosmetic effects of the ancient formula which claimed whitening and smoothening effects, it is envisaged that the following procedures be taken: i. Proper authentication of the selected herbs; ii. Three formulae of slightly different herbal combination will be prepared to be tested in the same platforms to achieve comparative effects; iii. Whitening effects will be studied using melanocyte in vitro cultures together with collagenase enzyme inhibition tests and tyrosinase activity assay; iv. Skin penetration is essential for the topical application of any topical agent; hence ex vivo tests using porcine skin and diffusion cell system are to be performed; v. Finally, in vivo experiments using artificially induced skin melanin pigmentations in C57Bl/6 mice and topical applications of the formulae will be performed to compare the cosmetic effects; vi. The final outcomes would reveal a favourable formula and evidences of its cosmetic effects. Materials and Methods Herbal materials authentication and preparation Herbal material authentication Raw herbal material of Atractylodis Macrocephalae Rhizoma, Poria, Angelicae Dahuricae Radix, Paeoniae Alba Radix, Ampelopsis Radix, Bombyx Batryticatus, Tribuli Fructus, and Bletillae Rhizoma were purchased from a renowned supplier in Hong Kong. All herbs were chemically authenticated using Thin Layer Chromatography (TLC) in accordance to Chinese Pharmacopoeia (CP) [10]. Upon chemical authentication, herbarium voucher specimens of Atractylodis Macrocephalae Rhizoma (20153453), Poria (2015-3454), Angelicae Dahuricae Radix (2015-3457), Paeoniae. Alba Radix (2015-3455), Ampelopsis Radix (2015-3456), Bombyx Batryticatus (20153459), Tribuli Fructus (2015-3458) and Bletillae Rhizoma (2015-3460) were deposited at the museum of the Institute of Chinese Medicine at the Chinese University of Hong Kong (CUHK). Herbal extract preparation Extractions of herbs were performed following the traditional practice of herbal extraction. For each formula, all raw herbs were mixed in the same ratio and extracted twice by boiling under reflux at 100 °C using 10x distilled water. The aqueous extracts were then combined and filtered using cotton wool and concentrated under reduced pressure at 60 °C. The concentrated extracts were freezedried and the yield were recorded. All the extracts were stored in desiccators at room temperature before use. In vitro cell culture experiments Cell culture: The B16 melanoma cell line was obtained from the American Type Culture Collection CRL-6322 (USA). Cells were grown and maintained in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% (v/v) fetal bovine serum (Gibco, USA), 100 U/ml penicillin, and 100 μg/ml streptomycin in a 10% CO2 humidified atmosphere at 37 °C. Cells grown to 80% confluence in T75 culture flasks were trypsinized and seeded into 12, 24or 96well culture plates for experiments. Cell viability assay: Briefly, using 96-well plate, B16 cells were seeded at 4 x 103 cells/well. Cells were then treated with various concentrations of aqueous herbal extracts in distilled water (0 1 mg/ml), with or without 10 nM α-Melanocyte-Stimulating Hormone (MSH)-induction for 72 hrs. The relative amount of viable cells were determined by measuring the reduction of MTT dye in live cells to blue formazan crystals at optical density at 540 nm (Sigma-Aldrich, USA). Cellular melanogenesis assay: The amount of melanin present in α-Melanocyte-Stimulating Hormone (MSH)-induced melanoma was used as an index for melanogenesis in the present study. B16 cells were seeded onto 24-well plate at the density of 5 x 104 cells/well. Cells were incubated with 10 nM α-MSH for 72 hrs with or without different concentrations of aqueous herbal extracts in distilled water (0 1 mg/ml). Cells were washed with Phosphate Buffered Saline (PBS), followed by trypsinization. Cells were solubilized in 200 μl of 1 N NaOH containing 10% Dimethyl Sulfoxide (DMSO) at 80 oC for 1 hr. The absorbance of all samples were measured at 490 nm [11,12]. 2.5 nM kojic acid was used as the positive control. Cellular tyrosinase activity assay: For cellular tyrosinase activity measurement, cellular tyrosinase activity was assayed in terms of DOPA oxidase activity. B16 cells were seeded onto 6-well plate at the density of 1.2 x 104 cells/well. Cells were incubated with 10 nM α-MSH for 72 hrs with or without different concentrations of aqueous herbal extracts prepared using distilled water (0 1 mg/ Citation: Wat E, Siu WS, Chan HY, Ka Tso TH, Law HW, et al. In Search of an Innovative Agent for Skin Care Putting an Ancient Herbal Cosmetic Formula on Modern Bioactivity Testing Platforms. J Clin Investigat Dermatol. 2019;7(1): 5 J Clin Investigat Dermatol 7(1): 5 (2019) Page 03 ISSN: 2373-1044 ml). Cells were sonicated with phosphate buffer (pH 6.8) containing 1 mM Phenylmethanesulfonyl Fluoride (PMSF) (Sigma-Aldrich, St. Louis, MO. USA.), followed by centrifugation at 10,000 x g. Cell lysate were mixed with 5 mM L-DOPA (Sigma-Aldrich, St. Louis, MO. USA.), followed by incubation at 37 oC for 1 hr, and absorbance was measured spectrophotometrically at 475 nm [13]. 2.5 nM kojic acid was used as the positive control. Collagenase activity assay: Collagenase inhibition was determined using the commercially available Collagenase Activity Assay Kit (Colorimetric) (Abcam, UK). Test samples were added to the reaction mixture, followed by addition of 0.35 unit/ml collagenase. Enzymatic activity was assessed by measuring the reduction at A345 spectrophotometrically for 20 mins. In our assay, 50 μM EGCG was used as positive control as previously described [14]. UV-irradiation animal study: Animals and diet: All experiments were carried out in accordance with the guidelines approved by the Animal Research Ethics Committee at the Chinese University of Hong Kong (CUHK) (AEEC approval no.: 16/174/MIS). Female C57Bl/6 mice (8-week old) were supplied by the Laboratory Animal Services Centre, the Chinese University of Hong Kong. All mice were housed in normal standard cages (5 animals per cage) at a constant temperature of 21