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Keywords:

  • antioxidant activity;
  • effective utilization;
  • scallop shells;
  • skin protection

Approximately 300 000 t/annum of scallop shells are generated as industrial waste in the Hokkaido area of Japan alone. About 98% of scallop shell is composed of calcium carbonate, and the organic components constitute only 2% of weight of the scallop shell. Because calcium carbonate is a very cheap material, it is necessary to find a characteristic quality of scallop shells in order to effectively utilize this natural material. Calcium oxide after calcination of scallop shells have previously been used as an antibacterial agent,1 a food supplement, and an adsorbent of formaldehyde. The authors have previously shown that the organic components from scallop shell (scallop shell extract) enhance the turnover rate of the skin epidermal layer and increase the rate of recovery of UV-induced injury to rat dorsal skin.2 This property of scallop shell extract suggested that it may be suitable as a cosmetic material.3 The authors also reasoned that scallop shell extract may possess both growth-promoting activity for keratinocyte cells and antioxidative activity.2,3

Squalene monohydroperoxide is a primary oxidized lipid produced by UV radiation of skin.4,5 The generation of squalene monohydroperoxide has been reported in the pathogenesis of several skin conditions including acne6 and sunburn.7 The formation of squalene monohydroperoxide leads to peroxidation of cellular membranes, resulting in cell toxicity in vitro.8 Chiba et al. reported that topical application of squalene monohydroperoxide to hairless mouse skin induces wrinkle formation, roughness and water loss in skin.9 In addition, epidermal hyperplasia and dermal alterations such as collagen degradation and increased levels of glycosaminoglycan are induced in squalene monohydroperoxide-treated skin.10 These findings suggest that hairless mouse skin constitutes an effective model for studying wrinkle formation and skin aging in relation to oxidative stress in vivo.

In this present study, the effect of scallop shell extract on squalene monohydroperoxide-induced changes in rat dorsal skin was investigated. Scallop shell extract was prepared as described previously.1,2 Male Wistar rats were purchased from Hokudo (Sapporo, Japan) and fed a normal diet and water. Rats were cared for according to the Guidelines of Animal Care issued by the Office of the Prime Minister of Japan. Squalene monohydroperoxide was prepared as described by Chiba et al.9 Briefly, 5 mL of squalene solution (1 mg/mL) was UV-irradiated at 365 nm (0.24 J/cm2) for 1 h, and the solution was extracted twice with 5 mL of methanol. The methanol extract, containing squalene monohydroperoxide, was separated by reverse phase C18 column chromatography using 100% methanol, and the peak containing squalene monohydroperoxide was pooled. The solvent was evaporated under a stream of nitrogen gas and the squalene monohydroperoxide dissolved in ethanol to a final concentration of 12.3 mg/mL (dry weight/vol.).

The squalene monohydroperoxide solution (0.2 mL) or vehicle (0.2 mL of 100% ethanol) was applied to the shaved dorsal skin of 7 week-old rats once a day. After 1 week, changes in theskin surface were photographed using a digital camera (FinePix 1700z; Fuji Film, Tokyo, Japan). Application of squalene monohydroperoxide induced erythema and wrinkle formation on the dorsal skin (Fig. 1a). The surface of squalene monohydroperoxide-treated skin appeared to be rough when compared to that of vehicle-treated skin.9 These results suggest that the action of squalene monohydroperoxide is also observed in rat dorsal skin as well as in hairless mouse skin. To investigate the effect of scallop shell extract on squalene monohydroperoxide-induced changes in skin, the scallop shell extract was applied to the skin. After 4 h of the treatment with squalene monohydroperoxide solution, 0.2 mL of the scallop shell extract (5 mg/mL [dry weight/vol.]) or vehicle (water) was applied to the skin. Application of scallop shell extract inhibited erythema and wrinkle formation (Fig. 1b). Histological studies also revealed that the application of squalene monohydroperoxide increased thickening of the epidermal layer by 1.5 fold as described by Chiba et al.9 However, the epidermis thickening induced by squalene monohydroperoxide was inhibited by treatment with scallop shell extract (Fig. 2), suggesting that the scallop shell extract is effective against squalene monohydroperoxide-induced oxidative stress in the skin.

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Figure 1. Effect of the scallop shell extract on squalene monohydroperoxide-induced erythema and wrinkle formation in rat dorsal skin. (1) (a) Squalene monohydroperoxide or (b) vehicle (100% ethanol solution) was applied to the shaved rat dorsal skin once a day for a week. Arrow indicates wrinkle formation. (2) (a) Scallop shell extract or (b) vehicle (water) was applied to squalene monohydroperoxide-treated skin.

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Figure 2. Histological study of squalene monohydroperoxide-induced changes in skin. Cross-sections of formalin-fixed rat skin. The cross-sections of rat skin treated with (a) scallop shell extract or (b) vehicle (water) after the application of squalene monohydroperoxide were compared. Strips of skin were removed, fixed with 3.3% formalin and then stained with hematoxylin-eosin. (c) The thickness of skin epidermis was measured. Lane 1, untreated with squalene monohydroperoxide; lane 2, treated with squalene monohydroperoxide and vehicle; lane 3, treated with squalene monohydroperoxide and scallop shell extract. The thickness of skin epidermis was measured at eight points using NIH image software. Values represent the mean and standard deviations of measurements in three rats. Differences were determined by one-way anova. Subsequent analysis for significant differences between two groups was performed using the multiple comparison Student-Newman-Keuls test. *P < 0.05 relative to vehicle treatment.

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The mechanism of wrinkle and erythema formation by squalene monohydroperoxide is not clear. Reactive oxygen species production may be a common intermediate step in processes that initiate tissue damage leading to wrinkle formation. Squalene monohydroperoxide may produce free radical species during metabolism in skin. The authors previously reported the inhibitory activity on the peroxidation of linoleic acid and on production of superoxide anion which was generated by the reaction of xanthine and xanthine oxidase of scallop shell extract.2 The antioxidant ability of scallop shell extract appears to be effective at combating squalene monohydroperoxide-induced erythema and wrinkle formation. The results presented here suggest that scallop shell extract is effective against oxidative stress in skin.

Scallop shell extract includes unidentified proteins, glycoproteins, carotenoids, and organic compounds. Now it remains unclear whether bioactive substances are proteins or the other components. It will be necessary to isolate and identify these substances in future.

ACKNOWLEDGMENTS

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This work was partly supported by a Grant-in-Aid to Y Hasegawa from the Ministry of Education, Science, Sports and Culture of Japan.

REFERENCES

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  2. ACKNOWLEDGMENTS
  3. REFERENCES