The Effect of Asiaticoside on Guinea Pig Skin

Abstract

Asiaticosidet is a glycosidic terpene (1) obtained from the plant Centella asiatica reported to improve the healing of surface wounds (2-5). It is of interest to define more precisely its local biochemical and histological effects upon tissue, and to note possible local toxic effects. Tissue culture offers a convenient approach to this problem and methods for measuring the respiration of skin in vitro combined with histological study have been developed to provide information on the local toxicity of antibiotics, germicides and bacterial products (6-8). These technics were used to investigate the effect of asiaticoside upon skin, and to compare its local toxicity with that of other compounds. The effect of asiaticoside on the respiration of fresh slices of guinea pig skin was determined by the method of Lawrence (7), and a dose-response curve (Fig. 1) was constructed by plotting percentage respiratory survival of the skin after 24 hours against logarithms of the asiaticoside concentrations. The median effective concentration (ED50) can be calculated from this curve with some accuracy; the ED50 for asiaticoside was 2.9 mg/ml. Previous experience with antibiotics and germicides has shown that the ED30 (i.e., 30% respiratory damage) affords a critical level for comparison of toxicity because this is the maximum respiratory damage that skin can sustain consistent with migration of epithelial cells (7-8). This correlation has been confirmed with asiaticoside (9) at an ED30 of 1.3 mg/ml. Comparison with other therapeutic materials (8) suggests that asiaticoside is 2.6 times as toxic to skin as penicillin, a value comparable with many common antibiotics, e.g. chloramphenicol, erythromycin and neomycin. Experiments were also made to investigate the effect of asiaticoside in vivo. Guinea pigs were injected subcutaneously with 5 mg asiaticoside on one side and a second injection of the suspending fluid (saline) made on the opposite side, as a control. Erythema over the site of the asiaticoside injection was noted after half an hour, and by the second day there was evidence of a moderate local reaction. The control site appeared normal. The animals were killed two days after injection and specimens of skin removed from the injection sites for histological examination. Fig. 2 f Obtainable as “Madecassol,” Laroche-Navar-ronB Fig. 3 represents a section prepared from a comparable control injection. Asiaticoside induces heavy infiltration of macrophages into the dermis and all the biopsy specimens showed evidence of a fairly severe foreign body reaction. The epidermis also showed signs of disturbance in the region of the injection. The cells were swollen and stained abnormally, and the epidermis appeared thicker than normal in this region. This abnormality extended 2 to 3 millimeters from the point of injection, then gradually merged with normal epidermis. All sections prepared from control injection sites showed the histology of normal guinea pig skin. These experiments suggest that though local toxicity of asiaticoside is not great, certain concentrations have a direct effect on epidermal cells, which become swollen and distorted, causing an apparent increase in thickness of the epidermis. Asiaticoside produces similar histological effects on skin in vitro (9) and it was thought that the epidermal cells were becoming keratinized. There was no evidence in tissue culture of any increase in metabolic activity of skin even at concentrations much lower than those causing toxic effects. It is possible that asiaticoside might affect more E I .xz ~ t - sL I '<+ _ £*3oL <- S4ot - S 6cr o* y- i90r S9sL i_ I Q. 1 1 1__________I__________I__________I----------------- O 02 04 0-6 OS IOOO 1-2 Log Dose (mg/ml) Fig. 1. Dose-response graph showing the effect of asiaticoside on the respiration of guinea pig skin. Three separate experiments were made and each point is based on at least four observations. The limits of the highest and lowest observation are indicated. 95 96 THE JOURNAL OF INVESTIGATIVE DERMATOLOGY Fig. 2. Section of guinea pig skin two days after subcutaneous injection with asiaticoside. Hematoxylin and eosin. Fig. 3. Section of guinea pig skin two days after control injection. Hematoxylin and eosin. specific aspects of skin biochemistry, such as the formation of ground substance. Experiments concerning this, and the effect of asiaticoside on the healing of experimental burns in mice, will be reported shortly.