In vivo pro-angiogenic effects of dracorhodin perchlorate in zebrafish embryos: A novel bioactivity evaluation platform for commercial dragon blood samples

Abstract

Dragon blood has been used in wound treatment for many years and can be obtained from several distinct plant species. Dracorhodin, the active substituent of dragon blood, is a characteristic compound of the palm tree, Daemonorops draco. At present, the only method to evaluate the quality of commercial dragon blood samples is a HPLC method which determines the amount of dracorhodin in a dragon blood sample. In this study, we used zebrafish embryos as a platform to demonstrate the in vivo pro-angiogenic activity of dracorhodin perchlorate, the chemically synthesized analog of dracorhodin. By using this platform, three different commercial dragon blood samples were also examined. Our results clearly show that even though the commercial dragon blood samples had similar amounts of dracorhodin, they showed highly variable biological activity, such as pro-angiogenic effects and toxicity. In short, an in vivo activity assay platform for rapidly examining the biological activity of commercial dragon blood samples was successfully established here, which complements the current HPLC-based assay method.

Keywords: Angiogenesis; Dracorhodin chlorate; Dragon blood.

Fig. 1

Appearance of powder form and block form dragon blood. Block form dragon blood is made by mixing red tree resin with dammar gum. Powder form dragon blood (Sample A and B). Block form dragon blood (Sample C, D, E, and F).

Fig. 2

Effect of Dracorhodin perchlorate on sub-intestinal veins of zebrafish embryos. (A) Time line of zebrafish embryonic development showing the experimental procedures at different time points. (B) Sub-intestinal veins (SIV) of control embryo treated with 0.1% DMSO. (C–F) SIV of 72 hpf zebrafish embryos treated with (C) 0.31, (D) 0.62, (E) 1.25, and (F) 2.5 μg/ml of dracorhodin perchlorate (DP). White arrows indicate extra sprouts. (G) Quantification of average sprout numbers. DP increased the average sprout number in a dose-dependent manner between 0.31 and 1.25 and plateaued at 1.25–2.5 μg/ml. (H) Quantification of relative fold changes in sprout length with respect to controls. DP increases the sprout length in a dose-dependent manner. Data is expressed as a mean ± SEM from three independent experiments. Asterisks indicates P < 0.05 compared with the control group.

Fig. 3

Toxicity of crude dragon blood extracts on zebrafish embryos. Lethality of embryos at 72 hpf treated by DP or crude extracts of sample A, B and C. Data is expressed as a mean ± SEM from three independent experiments. *P < 0.05 compared with 0.1% DMSO treated controls.

Fig. 4

Comparisons of vascular phenotypes between commercially available dragon blood and DP. (A) SIV of an embryo treated with 0.1% DMSO. (B) SIV of an embryo treated with 1.25 μg/ml DP. (C) SIV of an embryo treated with a crude extract of Sample A containing 1.25 μg/ml dracorhodin. (D) SIV of an embryo treated with a crude extract of Sample C containing 1.25 μg/ml dracorhodin. (E) Quantification of average sprout number. (F) Relative fold changes in the sprout length with respect to controls. White arrows indicate extra sprouts. Quantification of vascular phenotypes expressed as a mean ± SEM. Asterisk indicates P < 0.05 compared with the control group.