Investigation of anti-proliferative and anti-angiogenic properties of Parkia javanica bark and fruit extracts in zebrafish

Abstract

The use of herbal products as traditional medicines has been a practice in India for centuries. Due to high ethnic diversity, the pool of herbal medicines is enormous, and they are often preferred over modern medicines in certain parts of the country. Cancer is one of the major non-communicable diseases affecting people worldwide. Despite considerable research, cancer is a disease that is still not understood completely, and there have been constant efforts towards the identification of novel drugs or approaches in cancer management. Parkia javanica, an important medicinal plant and a rich source of flavonoids and terpenoids, is widely studied for its antioxidant and anti-inflammatory activities. Traditionally, the fruit and bark extracts of P. javanica find use as home remedy for dysentery and piles in NE India. Moreover, the fruits are consumed by the people of North-East (NE) India as vegetables, either in steamed or cooked form. In this study, crude extracts of P. javanica fruit and bark were obtained, the sub-lethal dose was determined and were then analyzed for anti-proliferative and anti-angiogenic properties using a battery of assays in zebrafish embryos. The sub-lethal concentration 50 (LC50) was found to be 28.66 mg/L and 346.66 mg/L for bark and fruit extract respectively, indicating a decreased toxicity of the fruit extract compared to that of the bark. The anti-proliferative and anti-angiogenic properties were more pronounced for the fruit extract compared to the bark extract. Although preliminary, the results of the study suggest that P. javanica fruits possess potent anti-angiogenic and anti-proliferative properties, which can be further studied for the isolation of active phytochemicals for use as therapeutic agents.

Fig 1. Effect of Parkia javanica bark and fruit extracts on developmental toxicity.

(A) The PJ bark extracts showing morphological defects with 1/10^th concentration (2.86 ppm) of LC₅₀ compared to control group at different days of post fertilization. (B) & (C) represent the morphological defects of embryos treated with PJ fruit extract at 1/10^th and 1/100^th concentration of LC 50 i.e. 34.6 ppm and 3.46 ppm. (D) The cardiotoxicity of PJ fruit and bark in both control and treated groups was presented in (a) & (b), whereas the hatching ability was presented in (c) & (d). Asterisk indicates p-value <0.05.

Fig 2. Anti-angiogenic effects of Parkia javanica bark and fruit extract on zebrafish transgenic line Tg(kdrl:GFP).

(A) The anti-angiogenic activity of PJ bark extract in transgenic Tg(kdrl:GFP) line with 2.86 ppm at 48 hpf. (B) Graph depicting the CTCF values for PJ bark extract exposed to Tg(kdrl;GFP) embryos, identifying the intra segmental vessels at 48 hpf. (C) The effect of PJ bark extract (2.86 ppm) on Tg(kdrl:GFP) model in SIV basket at 72hpf. (D) The CTCF values for PJ bark extract exposed to Tg(Kdrl;GFP) embryos, identifying the intestinal basket at 72 hpf. (E) Representative images of Tg(kdrl:GFP) trunk ISVs (a: control; b: treated) with 3.46 ppm PJ fruit extract at 48 hpf, (F) Graph depicting the CTCF values for PJ fruit extract exposed to Tg(Kdrl;GFP) embryos, identifying the intra segmental vessels at 48 hpf. Asterisk indicates p-value <0.05.

Fig 3. Anti-proliferative effects of Parkia javanica bark and fruit extract on zebrafish.

(A) The number of cells represented in brown dots has decreased in embryos treated with fruit extract compared to the control in 1/10^th of LC₅₀ concentration (34.6 ppm). (B) The number of cells represented in brown dots in embryos treated with bark extract with 1/10^th concentration (2.86 ppm) did not show any difference compared to control.

Fig 4. Pro-apoptotic effect of P. javanica bark and fruit extracts.

(A) Representative images of embryos exposed to UV and treated with 2.86 ppm of bark extract showing pro-apoptosis compared to untreated embryos. (B) Graph showing the total cell fluoresce intensity exposed to 2.86 ppm PJ bark extract at 48 hpf embryos. (C) The embryos exposed to UV and treated with 3.46 ppm of fruit extract showing pro-apoptosis compared to untreated embryos. (D) Graph showing corrected total cell fluoresce of 48 hpf embryos exposed to 3.46 ppm PJ bark extract. Asterisk indicates p-value <0.05.