Study of the potential adverse effects caused by the dermal application of Dillenia indica L. fruit extract standardized to betulinic acid in rodents

Abstract

This study aimed to evaluate the potential adverse effects of the dermal administration of Dillenia indica Linnaeus (D. indica) fruit extract in healthy rodents; the extract was standardized to betulinic acid. In the initial phase, the acute effects were evaluated on the skin application site of a single extract dose. A skin irritation test was performed in male Wistar rats (n = 8/group) receiving the extract (50-150 mg/mL) with betulinic acid (0.5-1.5%, respectively). A photosensitivity test was performed in male BALB/c mice (n = 6/group) receiving the extract (150 mg/mL). Afterwards, other BALB/c mice (n = 20, male:female, 1:1) were used to assess the systemic alterations caused by 14 daily repeated doses (150 mg/mL) by monitoring the effects on mortality, body morphology, behavior, nutrition status, neuromotor reactions, organ morphology and weight, and blood tests. At this time, 0.5 mg/mL clobetasol was used as the positive control. The skin irritation index suggested that negligible skin irritation had occurred, even when the extract was applied to the rat skin at 150 mg/mL. However, the extract acted as a photosensitizer on mouse skin, showing a photosensitizing activity close to that of 10 mg/mL 5-methoxypsoralen. Repeated doses caused no mouse mortality, aggressiveness, piloerection, diarrhea, convulsions, neuromotor alterations or nutrition status changes. The mouse organ weights did not change, and the mice did not have alterations in their blood compositions. Clobetasol caused a reduction in the mononuclear leukocyte numbers. In general, the data suggest that the extract was safe in healthy rodents but indicate that caution should be taken with the photosensitizing activity; in addition, this activity should be further explored as it may be useful for phototherapeutic drug development.

Fig 1. Study design for evaluating the potential adverse effects caused by Dillenia indica L. fruit extract standardized to betulinic acid and dermally applied to healthy rodents.

Fig 2. Evaluation data of the photosensitivity-inducing potential of standardized Dillenia indica fruit extract (1.5% betulinic acid) (single dose) dermally administered at 150 mg/mL (100 μL) on a shaved area of the back of healthy male BALB/c mice (n = 6/group).

Only vehicle (ethanol:water 1:5, 100 μL) was administered to the mice from the untreated and the vehicle control groups, whereas 10 mg/mL 5-methoxypsoralen was applied to the mice in the positive control group. Thirty minutes later, the skin application sites were irradiated with ultraviolet light at a sufficient intensity to cause minimal erythema (1.5 J/cm² at 20 cm for 20 min). Any erythema increase was evaluated 24 h after irradiation. Photosensitization was evaluated with infrared thermography (data in B) performed in the same skin area (as that shown in A) and was evaluated by erythema scoring (data in C). The data were analyzed by the analysis of variance (ANOVA) and the Bonferroni test. *denotes a significant difference in comparison to the vehicle control group (p < 0.05).

Fig 3

Number or crossovers representing the average ambulation behavior (A) and grip force (B) of mice from both sexes. The mice were healthy BALB/c mice (n = 20, male:female 1:1), which were dermally treated every 24 h for 14 consecutive days on an intact skin area (4 cm²) on their backs. One group received standardized Dillenia indica fruit extract (1.5% betulinic acid) at 150 mg/mL (100 μL), whereas 0.5 mg/mL clobetasol was administered to the mice from the positive control group. Only the vehicle (100 μL ethanol:water 1:5) was administered to the mice from the vehicle control group. The data were analyzed by analysis of variance (ANOVA). No significant differences were verified among the data.