Bioactivity Study of Barringtonia asiatica (Linnaeus) Kurz. Seed Aqueous Extract in Artemia salina

MATERIALS AND METHODS

Extraction: Mature fruits of Barringtonia asiatica were collected from Infanta, Quezon, Philippines on May 2005. The seeds were removed from the fibrous mesocarp and the brown seed coating. It was then grated, packed in aluminum foil and stored in the freezer ready for extraction.

The extraction of the plant material was done by soaking the grated sample in distilled water for 24 h. Crude extract was obtained by filtration and further concentrated by using freeze drier. The collected brownish extract was lyophilized to obtain powdered form of the extracts.

Biological assay: The tests were conducted in 96-well microtiter plate (final volume of 200 μL) using sterilized seawater. A stock solution of the crude extract (10,000 ppm) was prepared using artificial sea water. Different concentrations of extracts ranging from 1-500 ppm were prepared from the stock solution. Controls were also prepared and used with distilled sea water as negative control and potassium dichromate as positive standard. One hundred microliter of each working solutions of the extract was transferred in an individual well of a 96-well microtiter plate.

Brine shrimp hatchability test: The brine shrimp hatchability test is based on the procedure done by Migliore et al. (1997). Dried cysts of about 100 mg were allowed to hatch in 100 mL seawater at 28°C, under conditions of continuous illumination and strong aeration. After 2 h, 50 μL aliquots were placed in each well where the extracts had previously been deposited. They were incubated at the same conditions of temperature and illumination under gentle shaking. The free nauplii in each well were counted under a stereoscopic microscope after 12, 24 and 48 h of exposure. Five replicates were used for each treatment and control. The percentages of hatchability were calculated by comparing the number of free nauplii in each treatment with the number of free nauplii in the control. Later the percentage of hatch inhibition (HI%) was calculated as:

Brine shrimp lethality test: Dried cysts were incubated (100 mg cyst per 100 mL) in a hatcher at 28-30°C with strong aeration, under a continuous light regime. Phototropic nauplii were collected with a micropipette from the lighted side and placed in a small vial 12 h after hatching. Ten brine shrimps were placed in each well which contained the extracts previously deposited. These brine shrimps were exposed to various concentrations of the extract. The mortality was determined after 12 h (mainly nauplii in instar I/II), 24 h (nauplii in instar II/III) and 48 h (mainly nauplii in instar III/IV) of exposure. The larvae did not receive food. To make sure that the mortality observed in the bioassay could be attributed to bioactive compounds and not to starvation, the number of dead larvae in each treatment were compared to the number of dead larvae in the control. In any case, hatched brine shrimp nauplii can survive for up to 48 h without food (Lewis, 1995) because they still feed on their yolk-sac (Pelka et al., 2000). However, in cases where control deaths were detected, the percentage of mortality (% M) was calculated as:

The number of survivors was counted under a microscope and percentage of mortality was calculated. Larvae were considered dead if they did not show signs of any internal or external movement during several seconds of observation under a microscope.

Phytochemical test: The presence of saponins and terpenoids in the extract was determined using the froth test, hemolytic assay and Liebermann-Burchard test for saponins and the Salkowski test for terpenoids. Froth test was done wherein 2 g of the powdered sample was boiled in 20 mL of distilled water in a water bath and filtered (Oboh et al., 1999). The filtrate (5 mL) was mixed with 5 mL distilled water and shaken vigorously for a stable persistent froth. On the other hand, hemolytic assay was done by mixing the crude extract with red blood cell solution in microplate wells using the method by Mojica and Merca (2005). Lastly, for the Liebermann-Burchard test, 10 drops of the crude extract was placed in dry test tubes. It was added with 3 drops of acetic anhydride and one drop of concentrated sulfuric acid and observed for any color changes (Said et al., 1990).

For the test for terpenoids, 5 mL of the crude extract was mixed with 2 mL of chloroform and concentrated sulfuric acid (3 mL) was carefully added to form a layer.

RESULTS AND DISCUSSION

Brine shrimp hatchability and lethality test assay were used to determine the biological activity of the Barringtonia asiatica (botong) seed’s aqueous extract. For the hatchability test, inhibition of hatching was observed in at least 10 ppm extract for 12 h and 100 ppm for 24 h exposure (Fig. 1). At lower concentrations (1-50 ppm), there is a dose dependent relationship wherein the percentage hatchability decreases as the concentration of the extract increases. The low hatching rate observed after the 12 h treatment was probably due to an alteration in the development of Artemia embryos. Studies have shown that Artemia is highly vulnerable to toxins at the early developmental stages (Sorgeloos et al., 1978; Sleet and Brendel, 1985). The hatched brine shrimp nauplii in treated with the crude extract were found to have deformities just like those observed in the positive control in comparison to the nauplii in the negative control.

In the lethality/mortality test, the same dose dependent relationship just like the hatchability test was observed wherein the percentage mortality increases as the concentration of the extract increased (Fig. 2). The LC₅₀ was found to be 275.7, 32.34 and 2.25 ppm for 12, 24 and 48 h of exposure, respectively. This is higher in comparison to the positive control which have LC₅₀ of510.4, 139.4 and 20.25 ppm for 12, 24 and 48 h of exposure, respectively. Activity increased significantly up to 48 h exposure particularly for the fruit extract. Maximum sensibility is usually reached after 48 h of exposure (the oldest age class tested) (Sanchez-Fortun et al., 1996) since at this stage, the life cycle of the nauplii have reached second and third instar and exhibit greatest sensitivity to test compounds (Lewis, 1995).

Fig. 1:	Artemia salina (brine shrimp) hatchability test of Barringtonia asiatica crude water extract. Percentage hatchability of brine shrimp eggs was monitored after 12 and 24 h exposure on different concentrations of the extracts

Fig. 2:	Artemia salina (brine shrimp) lethality test of Barringtonia asiatica crude water extract. Percentage mortality of brine shrimp eggs was monitored after 12, 24 and 48 h exposure on different concentrations of the extracts

For the past 30 years, the Artemia nauplii have been used detect general toxicity (Persoone and Wells, 1987), in teratology screens (Acey and Tomlison, 1988; Sleet and Brendel, 1985) and in ecotoxicology (Sorgeloos et al., 1978). From a pharmacological point of view, a good relationship has been found with the brine shrimp lethality test to detect antitumoral compounds in terrestrial plant extracts (Meyer et al., 1982; Solis et al., 1993). The extract could contain antitumor agents since the LC obtained is lower than the standard set by the United States National Cancer Institute. For a bioactive compound to be an effective antitumor agent it must have an LC₅₀ equal or less than 30 ppm. This is also more potent than the previous study done on bignay [Antidesma bunius (L.) Spreng] which have an LC₅₀ of 702.13 ppm for 12, 24 and 48 h of exposure, respectively (Micor et al., 2005). With these preliminary results, it is possible that the Barringtonia asiatica contain substances that might have cytotoxic activity.

For the phytochemical tests conducted, the crude extracts gave positive results for the presence of saponinss. Froth test showed persistent honeycomb frothings on the extract and Leibermann-Buchard test showed the formation of a greenish color in the solution. Positive results were also obtained in the hemolytic test thus confirming the presence of saponins. On the other hand, a reddish brown coloration on the interphase was obtained in the Salkowski test confirming the presence of trepenoids. Literature had reported the presence of these phytochemicals (Herlt et al., 2002; Burton et al., 2003; Yang et al., 2006) in the seeds of Barringtonia asiatica and its related genus. It is possible that these compounds acting individually or synergistically were responsible for the observed activity of the extract.

CONCLUSION

Aqueous crude extract of Barringtonia asiatica seeds were tested for biological activity using the brine shrimp hatchability assay and lethality assay. Results showed dose dependent relationship on the activity and concentrations used. It is possible that the extracts contained substances with cytotoxic activity. Phytochemical tests using the crude extracts showed the presence of saponins and terpenoids.

ACKNOWLEDGMENTS

We would like to acknowledge Dr. Florinia E. Merca of the Institute of Chemistry, University of the Philippines Los Baños (UPLB) for the botong samples and Dr. Custer C. Deocaris of the National Institute of Advanced Industrial Science and Technology (AIST) based in Tsukuba, Japan for the suggestions in the modification of the brine shrimp assays.