ANTIBACTERIAL ACTIVITY AND THE MECHANISM OF ACTION OF EXTRACT, FRACTION AND SUBFRACTION OF PICUNG SEEDS (Pangium edule Reinw.)

• Main Author: Saptarini, Opstaria
• Format: Dissertations
• Language: Indonesia
• Online Access: https://digilib.itb.ac.id/gdl/view/36790
• Institution: Institut Teknologi Bandung

Infectious disease is one major issues in developing countries. In Indonesia. It is a main contributor to mortalitiy and morbidity in hospitlas and other health service centers. Based on recent results of basic health research, the prevalence of infectious disease in Indonesia is still high as shown by the rates of pneumonia, hepatitis and diarrhea which account for 1.8, 4.5 and 7.0%, respectively. Infectious disease is generally treated with antibiotic. Studies have revealed 40-62% of antibiotic inappropriate use, including overuse and un-indicated use of antibiotics, which could lead to microbial resistance. This poses challenges to the invention of alternatives to fight microbial infections and natural product, including medicinal plants, could be the source for the development of antibacterial substances. Picung (Pangium edule. Reinw) is a plant that generally grows on the banks of rivers, watery areas, primary forests, secondary forests, and community gardens. Picung is a native plant that grows in Southeast Asia and has been empirically used to preserve fish. In addition, Picung in the form of extract has been investigated on its ability to inhibit the growth of both Gram-positive and Gram-negative bacteria, including Micrococcus luteus, Staphylococcus aureus, Alcaligenes eutrophus, Escherichia coli and Enterobacter aerogenes. Picung is known to have high cyanide acid content, both in the stem, leaves, and fruit, but this toxic ingredient is easily removed because it is soluble in water and will be evaporate at 26 ° C with washing. The flesh of picung seeds contains alkaloids, flavonoids, terpenoids/steriods and polyphenols. In this research the antibacterial activity of seeds extracts and fractions of Picung were tested against the Gram-positive bacteria, S. aureus, MRS and Bacillus subtilis; and the Gram-negative bacteria, Pseudomonas aeruginosa, E. coli and Salmonella typhi. The sites of action of the active farction and sub-fraction of the Picung seeds were tested based on the results of leak test and SEM and TEM examinations. In addition, the safety aspect of ethanol extract of Picung seeds was also investigated. The raw drug of Picung was collected from Bogor Tropical Plant Research Center (Balitro). The ethanol extract was prepared by refluxing the raw drug with 96% ethanol. The extract was added with hot water and immediately filtered. The filtrate was fractionated by liquid-liquid extraction method using solvents of different polarity, to obtain three fractions, whichn-hexane, ethyl acetate and water fractions. The extract and fractions of Picung seeds were tested for their antibacterial activity with diffusion method followed by microdilution against the growth the Gram-positive bacteri S. aureus (ATCC 6538), Methicillin-Resistant Staphylococcus aureus (MRSA), B. subtilis (ATCC 6633); and the Gram-negative bacteria P. aeruginosa (ATCC 9027), E. coli (ATCC 8939), and S. typhi. Antibacterial activity is determined based on the minimum inhibitory concentration (MIC), compared with the reference antibiotics amoxicillin and tetracycline. Preliminary results showed that the most active fraction was ethyl acetate fraction against S. aureus and E. coli. This active fraction was then subject to separation using vacuum column chromatography to obtain ethyl acetate subfraction. The fractions were then tested for the sites of action on the bacteria by performing leak test, and microscopic examination using SEM and TEM. Time-kill curve was also observed to determine bactericidal and bacteriostatic property. The safety test of Picung ethanol extract was carried out by acute toxicity test in female mice. The mice were initially divided into groups, each to receive 0.1% CMC, low concentration of extract, 1000 mg/kg, and 5000 mg/kg extract. Due to the absence of signs of toxicity, two groups of mice were added to receive 10,000 mg/kg and 15,000 mg/kg extract, respectively. The behavior and mortality were were assessed during a 14-day period of observation. LD50 was calculated using probit method and changes in body weight were recorded. Phytochemical screening revealed positive result for flavonoid in extract and fractions, except for n-hexane fraction. Positive result for alkaloid was found in extract and fraction, except for water fraction. Extract and all fractions were positive for steroid/triterpenoid. Thin layer chromatogram indicated the presence of steroid/triterpenoid in extract and n-hexane as well as ethyl acetate fractions. Meanwhile, the chromatogram of ethyl acetate subfraction obtained from vacuum liquid chromatograpy revealed two spots, suggested to contain stigmasterol and fatty acid. Results of antibacterial acitivity by diffusion method against S. aureus, MRSA, B. subtilis, P. aeruginosa, E. coli, and S. typhi showed that ethyl acetate fraction was the most potent at concentration of 1%. By microdilution method, it was shown that ethyl acetate fraction of Picung seeds was more potent than other fraction in inhibiting bacterial growth with MIC between 256-512 ug/ml, albeit less potent than the reference drug amoxycillin and tetracyclin with respective MIC of 1 µg/ml. Antibacterial activity tests of active subfraction of Picung seed showed that the subfraction was active against S. aureus, E. coli and P. aeruginosa, but with the conncentration of 1% the highest activity was shown against E. coli with the highest inhibition zone of 11 mm. Observation on toxicity signs was carried out intensively during the first hours and continued through 24 hours after administration of test substances. It was found that extract of Picung seeds did not induce abnormal behaviors and death in all treatment groups. Furthermore, no changes in body were found during the observation period. SEM examination showed morphological changes on E. coli and S. aureus cell walls upon exposure to 512 mg/ml of active fraction and 1024 mg/ml of active subfraction. This suggests inhibition of cell wall biosynthesis leading to its damage. This was further confirmed by observation using TEM which revealed cell lysis upon exposure to 512 ug/ml of active fraction and 1024 ug/ml active subfraction in S. aureus. Increased release of cellular contents upon measurement corroborate the cytolytic activity of the fraction and subfraction. The results of this study concluded that ethanol extract, ethyl acetate fraction and picung seed subfraction had antibacterial activity against Gram positive and Gram negative bacteria. The active fraction and subfraction of picung seeds based on the results of observations using the SEM and TEM tools can be seen that the location of work is on bacterial cell walls, because it can cause disruption of permeability and damage (lysis) on bacterial cell walls, and this is confirmed by membrane leak testing shows an increase in the number of proteins and nucleic acids from bacterial cells in the supernatant. The results of acute toxicity testing of the picung ethanol extract were non-toxic / safe against test animals