Here, we present the protocol for using ethanol as a solvent to extract and characterize Taiwanese green propolis which inhibit antibacterial activity. This protocol is a reliable and repeatable method for determining the quality of Taiwanese green propolis. Demonstrating procedure will be Chun'Ting Chen and Yi'Hsuan Chien, PhD students from my laboratory.
To begin, weigh out 100 grams of Taiwanese green propolis. Using a spice grinder, grind the propolis making sure that the pieces are ground into a fine powder without any large particles. Set out five flasks and add 100 milliliters of various concentrations of ethanol in water to each.
Mix 10 grams of ground propolis into the ethanol solution in each flask, and incubate at 25 degrees Celsius with shaking at 250 RPM for 48 hours. After this, use filter paper with a 25 micrometer pore size to filter the ethanol extracts. Using a volumetric flask, reconstitute the filtrates to their original volume of 100 milliliters with 95%ethanol.
Store the reconstituted extracts at minus 20 degrees Celsius until ready to use. When ready to prepare the extracts for HPLC, use vacuum evaporation to concentrate 10 milliliters of each extract at 40 degrees Celsius for 10 minutes. Bake the dry matter at 45 degrees Celsius for 24 hours.
Then, reconstitute each sample of dry matter with 10 milliliters of 95%ethanol. Using a syringe filter with a 0.22 micrometer pore size, refilter each extract and collect the filtrate for direct analysis with HPLC. To begin establishing a standard curve, prepare one liter of the mobile phase methanol to water solution at a ratio of 88.8 to 11.2.
Using the mobile phase solution as a solvent, prepare serial dilutions of the propolin standard concentrations as outlined in the text protocol. Inject 20 microliters of each standard concentration into the reverse phase columns sequentially from the lowest concentration to the highest. Then, set the HPLC column to 30 degrees Celsius, set the waive length of the UV detector to 280 nanometers, and the flow rate to one milliliter per minute, and the recorder time to 20 minutes.
Analyze the standards at least three times. After this, use calculation sheet software to plug the measurement response against the concentration resulting in a standard curve with a determined equation and R-squared value. To begin analyzing the extracts, inject 20 microliters of each extract into the reverse phase column.
Set the HPLC column to a temperature of 30 degrees Celsius, and a flow rate of one milliliter per minute. Set the waive length of the UV detector to 280 nanometers, and the recorder time to 20 minutes. Analyze the extracts at least three times.
Then, use the standard curve to determine the concentration of propolin in each ethanol extract. After preparing the test organisms and ethanol extracts, add 10 microliters of diluted ethanol extract, ranging from 0.156 to 640 micrograms per milliliter into the wells of a 96 well plate. Use broth to adjust the volume in each well to 100 microliters making sure to maintain 5%DMSO in all the dilutions.
Next, inoculate 100 microliters of bacterial culture into each well of the 96 well plate. Culture at 37 degrees Celsius for 48 hours. Use a microplate reader at 590 nanometers to analyze the bacterial growth according to turbidity and to determine the minimum inhibitory concentration.
After this, inoculate 10 microliters of liquid culture from each well that exhibited to growth onto an agar plate. Incubate at 37 degrees Celsius for 24 hours. Identify the lowest concentration that revealed no visible bacterial growth to determine the bacterial activity.
The concentration that completely eliminated cell growth is considered to be the minimum bacterial concentration. In this study, Taiwanese green propolis is extracted with ethanol. The dry matter yield is seemed to be highest when a high concentration of ethanol is used, and lowest when water is used.
These results indicate that an organic solvent performs best during this extraction, and that the yield is positively associated with the ethanol concentration. Likewise, the concentration of propolins is seemed to be positively associated with the ethanol concentration during extraction, with the highest yield of propolins being obtained in the 95%and 99.5%ethanol extracts. The antibacterial affects of the ethanol extracts against the S aureus and E coli are then investigated.
The average minimum inhibitory concentration for S aureus is seemed to be between 10 and 20 micrograms per milliliter and the average bacterial concentration is seemed to be 20 micrograms per milliliter. Water extracts, however, did not have any antibacterial affect against S aureus. No antibacterial affect is observed for E coli when using either the water or ethanol extracts.
One of the key experimental procedure is eschewing the uniformity of Taiwanese green propolis during grinding.
