Cytotoxicity and Antioxidant Potentials of Barringtonia Asiatica Essential Oil

Right from ancient time drugs of plants have been used as herbal formulations in crude forms, like tinctures, teas, powders and poultices, for their growing interest as alternative therapies for the prevention or treatment of various diseases [1]. Essential oils from medicinal and aromatic plants are still considered as rich sources of a huge number of antimicrobial and antifungal components [2]. Many of them show a great potential as anticancer therapeutic agents [3]. However, essential oils are chemically characterized as complex mixtures of low molecular weight compounds and, some of them, are highly volatile and capable of generating flavors and aromas [4]. Scientific studies have shown the role of essential oils in biological interactions among plants and their potential therapeutic including anti-inflammatory, analgesic, anti-tumor, antifungal, and antibacterial activities [59]. In addition to the above-mentioned properties, many essential oils have been confirmed to possess antioxidant activity. They are extremely important for disease prevention, since they inhibit and delay the oxidation of biomolecules by preventing the initiation or propagation of chain oxidation reactions [10-12]. Thus, because of the harmful effects that synthetic antioxidants may cause, such as toxicity and carcinogenicity, the interest in the discovery of natural antioxidants has increased considerably [13]. The growing interest in natural bioactive compounds has led to conduct further studies addressing the replacement of synthetic chemical agents in the industrial sector, since natural products are less harmful to health [14], in addition to being biodegradable and usually exhibiting low toxicity in mammals [15]. This plant Barringtonia asiatica was considered to extract and test for its cytotoxicity and antioxidant potentials. However, Barringtonia asiatica is one of the species belonging to the family Barringtonia, native to mangrove habitats on the tropical, it is a common plant in the Malaysian Mangroves and wetlands such as the Kuching wetlands Sarawak and Bako National Park, it is also found in tropical Africa, Nigeria and Madagascar. Other species belonging to this genus are Barringtonia racemosa, Barrington acutangula, Barringtonia edilus, Barringtonia lanceolate, Barringtonia macrostachya, and Barringtonia spicata [16]. The literature survey revealed that there are no scientific Abstract

studies carried out regarding antioxidant and cytotoxicity of the essential oil of the leaves, stem-barks of Barringtonia asiatica.
Hence, the present study is focused to evaluate the antioxidant potentials including determining cytotoxicity (leafs and stembarks) by using Artemia salina and 2,2-diphenyl-1-picrylhydrazyl.

Extraction of Essential Oil
The plant samples were subjected to water distillation for 8 hours using Clevenger apparatus to extract the oils quantitatively, according to method described by Samsiah et al. [17] and Fasihuddin & Ibrahim [18]. Approximately 100 g of fresh cut and ground samples of Barringtonia asiatica leaf and stem bark were weighted, transferred into 2 L round flask and mixed with 1.35 L of distilled water. The flask was assembled to the Clevenger trap, connected to the condenser and heated. The sample was heated for 8 hours for hydro distillation process. After 8 hours, the oil trapped in the Clevenger was left to cool at room temperature. The water layer at the bottom of the oil was first drained out to separate it with the oil. The collected oily layer was treated with anhydrous sodium sulphate to remove any trace of water. The experiment was performed triplicates for each sample. The essential oil obtained was kept in a vial and stored in refrigerator at 4°C prior to analysis.

Brine Shrimp (Artemia salina) Lethality Test
Toxicity test against brine shrimp (Artemia salina) developed by McLaughlin et al. [19] was used in this study. Leached brine shrimp eggs were hatched in seawater and incubated for 48 hours at 25ºC. Exactly 3 mg of sample was dissolved in 3mL methanol, and the mixture was sonicated to ensure homogeneity of the extract.
Four different volumes of 500, 250, 50 and 5μL each from the stock solution were transferred into NUNC multidish in triplicate. Solvent was allowed to evaporate under a running fume hood for overnight and followed by the addition of 0.2mL DMSO and 4.8mL seawater to give final concentration of 100, 50, 10 and 1μg/mL, respectively.
Ten brine shrimp nauplii were transferred into each concentration in NUNC multidish, and was observed every 6 hours for 24 hours. The amount of dead nauplii were calculated. Thymol was used as positive control, whereas 0.2mL DMSO and 4.8mL seawater was used as negative control. The data was analysed to determine the concentration of the samples that kill 50% of brine shrimp at 24 hours or known as LC50. LC50 was calculated and determined by performing Probit analysis in IBM SPSS Statistic software of version 21.

Scavenging Assay
The free radical scavenging assay of compound 2,2-diphenyl-1-pycryl-hydrazyl (DPPH) was used to evaluate the antioxidant properties of the crude extract and the essential oil. The measurement was based on the method described by Wang et al. [20]. The sample was prepared by diluting 6 mg of crude extract into 6 mL of methanol, producing a concentration of 1000μg/mL.

Statistical Analysis
Statistical analysis for biological activities was performed using SPSS programme. The LC50 values for toxicity assay was calculated using the Probit Analysis option in the SPSS. The IC50 for DPPH free radical scavenging assay was statistically determined using Log dose inhibition curve in PRISM programme.

Results
The plant samples were subjected to water distillation for 8 hours using Clevenger apparatus to extract the oils quantitatively.

Discussion
The essential oil of the plant parts of Barringtonia asiatica tested showed a significant lervical activity when compared with the control and the report of [22]. The lethality concentration of the leaves and the stem-bark essential oil were observed at 1µg/mL -100 µg/mL as presented on the

Conclusion
This study showed that Barringtonia asiatica (L.) essential oil from leaves and Stem-bark have high antioxidant activity. Thus, further studies should be carried out to establish the bioactive compound, its application and practicability of incorporating Barringtonia asiatica (L.) essential oil in any healthy processed products such as capsulated daily supplements.