Clinical Test


Rozanida Abdul Rahman, Noorullhamezon Mohd Noor, Suzaini Badrudin, Harmayumi Wahid, Mohd Helme Mohd Helan, Jaafar Abdullah, Yap Say Moi, Hamidah Sidek

SIRIM Berhad, P.O. Box 7035, 40700 Shah Alam, Selangor, Malaysia

Keywords : fermentation, collagen synthesis, skin lightening, cosmeceutical

The cosmetic industry is constantly looking for substances able to preserve the skin in a healthy state and maintaining youthful appearance. The trend for natural and naturally-derived ingredient for cosmetic products continues to grow. Lately the use of food products such as enzymes and vinegars from herbal materials or fruits, and currently fermented filtrates has developed into an emerging source of new cosmeceutical actives. A fermented marine-based product has been investigated for its benefits to the skin. Similar marine-based fermented products from the Philippines and Japan has been reported to be a rich source of protein (Ernestina, M.P. et al., 2008). Fermentation transforms the organic substances into peptides and amino acids which not only contribute to the flavour and aroma but also as naturally occurring antioxidant. In our study, the activities of a fermented marine filtrate known as bioactive essence was investigated using ex-vivo and in vivo clinical tests. Ex-vivo test includes antioxidant, anti-melanogenesis, cell proliferation and collagen synthesis which are useful properties for the development of cosmeceutical products while in vivo clinical tests involves a placebo-controlled study on healthy Asian subjects.

Materials and Methods
Material. The traditionally fermented marine filtrate was processed by boiling, filtration and sterilization. The bioactive essence obtained was kept at 4ºC when not in use. Glutathione and total phenolic content were determined by glutathione assay kit and a modified Folin-Ciocalteu method (Yeh and Yen, 2005), respectively.
Antioxidant activity was measured by the free radical scavenging capacity assay using the stable DPPH (1,1-diphenyl-2-picryl-hydrazyl) radical according to the method by Hou et al., 2003 with ascorbic acid and α-tocopherol as positive controls.
Anti-melanogenisis activity was investigated by the tyrosinase inhibition assay using L-DOPA [3-(3,4 Dihydroxyphenyl-2,5,6)-L-alanine] as substrate with arbutin and kojic acid as positive control, while measurement of melanin content was measured on cultured melanocytes according to the method previously reported by Jones et al., 2002 with slight modification.

Proliferation and collagen synthesis in skin cells treated with the bioactive active essence was measured according to the methods by Kim et al., 2006 and Lee et al., 1998 with slight modification using cultured human fibroblasts. The collagen content in the fibroblast cell line treated with various concentrations of filtrate for 48h was measured by Sirius red method with human collagen type 1 as standard. The amount of collagen is expresses as micrograms per 100 μg protein.

Cytotoxicity evaluation was determined using the MTT [3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide] method according to the procedure of Mossman. Cells were plated in 96-well plates at 2 x 10^5 cells/well. After 48 h, test sample was added to each well and incubated for 48 h. Cell survival was determined in a colorimetric assay using mitochondrial dehydrogenase activity in active mitochondria to form purple formazan. Cell viability was calculated as follows: cell viability (%) = (absorbance of the sample tested/absorbance of the medium only) x 100.

Development of cosmeceuticals and clinical trials. The cosmeceutical products comprises of a Cleanser, Moisturiser and Serum with bioactive essence as the active ingredient. Clinical trials were conducted on a 24 panel healthy human subjects with skin phototypes III and IV in a placebo controlled double-blind randomised order. The products were applied to the face twice a day for 8 weeks and evaluations were made before and after treatment (Seong, Y.L., 2007). Objective instrumental measurements were carried out using the Corneometer® CM825 and Sebumeter® for moisturizing and sebum content, Mexameter® MX16 and Chromameter CR-10 for skin brightness and clarity and Cutometer® MPA 580 for skin elasticity and firmness. All the instruments are from Courage and Khazaka, Germany except for Chromameter (Minolta).

Results & Discussion
Free radicals induced by ultraviolet light or oxidative stress accelerate skin aging and can adversely affect skin health. The antioxidant activity of bioactive essence exhibited an IC50 value of 4mg/ml compared to ascorbic acid and α-tocopherol (IC50=0.012mg/ml; 0.0055mg/ml). The results imply that this bioactive essence may contain constituents with strong proton-donating capabilities to scavenge hydroxyl radicals and may protect cellular lipids against free radical reaction (Reiter et al., 2001). Bioactive essence also exhibited in vitro mushroom tyrosinase inhibition with IC50 value of 9 mg/ml comparable with arbutin at 10 mg/ml. The in-vitro free radical DPPH scavenging and mushroom tyrosinase inhibition effect is summarized in Table 1. Further evaluation on cellular melanin formation within the melanosome, did not show dose-dependent inhibition of melanin production (Figure 1). Treatment at concentration of 2.5 to 10mg/ml inhibited 60% melanin formation in B-16 melanocytes cell line with 100% cell viability.

Collagen is the predominant fibrous protein of the extra-cellular matrix and is a major protein constituting connective tissue in human body. About 3-6% of total tissue protein in the body is collagen, and the functional properties of skin depend on the integrity of collagen in the dermis. Changes in the rate of collagen deposition occur during wound healing, new bone development and with aging (Caradinale and Udenfriend, 1974). Therefore, the control of collagen metabolism may be useful for a variety of therapeutic and cosmetic applications. Figure 2 and 3 shows the effect of bioactive essence on skin rejuvenation in terms of promoting cell proliferation (up to 41% at 1.56mg/ml) and collagen synthesis (increase of 133 μg/100 μg protein at 0.625mg/ml).

The biological activities of bioactive essence are associated with the bioactive compounds present. Glutathione content and total phenolic compound in bioactive essence are 35μg/mL and 2.1mg/g GAE, respectively. Standard and well defined safety evaluation i.e. heavy metal tests, microbial count and toxicological studies demonstrated that the bioactive essence is safe to be used in cosmetic formulations (Table 2). In vivo product efficacy studies on 24 panel volunteers demonstrated significant (p<0.05) skin lightening effects (-7.68%), moisturizing (4.97 ± 2.98%) and improved skin elasticity (14.8 ± 0.1%) compared to control.

The fermented marine filtrate known as bioactive essence exhibited a wide range of biological activities such as antioxidant, cell proliferation, collagen synthesis and skin lightening properties and was found to be safe to be used in cosmetic formulations. Clinical trials studies have shown significant increase in skin moisturizing condition, skin brightness, clarity and firmness compared to control.

This work was supported financially by SIRIM Berhad Internal Fund and Ministry of Science & Technology Innovation (MOSTI). This work has been patented under invention titled: Fermented Marine-Based Material For Cosmeceutical Application.

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