Extraction and isolation of bioactive compound from piper betle leaves and antimicrobial efficacy of the emulsion

Abstract

Piper betle (daun sirih) extract and essential oil were reported to have multifunctional biological effects including medicinal and antimicrobial functions. The main objectives of this study were to extract bioactive compounds from the dried Piper betle leaves in order to determine the bioactive compounds from the extract, formulate the Piper betle emulsion, investigate the antibacterial activity and acute systemic toxicity of the Piper betle emulsion. The extraction of Piper betle leaves was prepared by using three extraction methods namely Soxhlet extraction method, supercritical carbon dioxide (SCO2) extraction method and probe type ultrasound - assisted extraction method. The probe type ultrasound - assisted extraction was found to be significant in improving extraction efficiency, reduce the consumption of extraction time and easy to be used. The extraction of Piper betle leaves using probe type ultrasound - assisted can achieve a high yield in a very short time (15 minutes) as compared to the Soxhlet extraction method (1440 minutes) and SCO2 extraction method (120 minutes). A total of 72 bioactive compounds from probe type ultrasound - assisted extraction and 84 bioactive compounds from SCO2 extraction were isolated and identified from the dried leaves of Piper betle by using gas chromatography - mass spectrometry (GC - MS) method. The presence of various bioactive compounds from Piper betle leaves justifies this plant contains medicinal properties and could be a promising source for potential antimicrobial agents. In this study, Piper betle emulsion was formulated using Piper betle essential oil, Tween 80, and water by using 20 kHz probe - type ultrasonic processor. Ultrasonic emulsification of Piper betle essential oil, Tween 80 and water was carried out and the effects of different concentration of surfactants (2.5 % w/v and 5 % w/v), various emulsification duration (30 minutes, 60 minutes and 90 minutes) and ultrasound intensity on the droplets size of emulsion has been studied. All of the parameters have significant effects on the diameter of droplets of Piper betle emulsions. It was observed that at 30 minutes of sonication time with surfactant concentration of 2.5 % w/v in 200 mL of water, a stable Piper betle emulsion with a mean diameter of droplet approximately 250 nm could be formed. The antibacterial activity of formulated emulsion was evaluated in - vitro and the minimal inhibitory concentration (MIC) was determined with the used of macrodilution method. The bacterial killing kinetic was also evaluated for this formulated emulsion. The MIC of the Piper betle emulsion for Streptococcus mutans was 2.5 mg/ml and Staphylococcus aureus was 5 mg/ml. The time kill kinetic study revealed that the formulated emulsion could act as microbiostatic agents. Meanwhile, the area under the curve for Piper betle emulsion against Streptococcus mutans and Staphylococcus aureus revealed that the number of bacterial cells were significantly (p < 0.05) reduced when compared to the control sample. This result proposed that, the formulated oil-in-water Piper betle emulsion has the potential to be used as an antibacterial agent against Streptococcus mutans and Staphylococcus aureus such as in the treatment of various oral diseases. Considering the pharmacological relevance of Piper betle leaves, the acute systemic toxic effects of the Piper betle emulsion were investigated. The overall findings of this study indicate that the Piper betle emulsion is non-toxic at the dose of 50 mL/kg body weight and can be considered safe for traditional and medicinal uses. From this study, the 20 kHz probe - type ultrasonic processor can be used to do the faster extraction of Piper betle leaves and form the oil-in-water Piper betle emulsion. The formulated Piper betle emulsion with mean diameter of 250 nm is confirmed non-toxic and can inhibit the growth of Streptococcus mutans and Staphylococcus aureus.