Extraction and Analysis of Taiwanese Green Propolis
January 7th, 2019
We present a protocol for using ethanol as a solvent to extract and characterize Taiwanese green propolis that exhibits antibacterial activity.
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.
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Overview
This article presents a protocol for using ethanol as a solvent to extract and characterize Taiwanese green propolis, which exhibits antibacterial activity. The method is reliable and repeatable, ensuring accurate quality determination of the propolis.
Key Study Components
Area of Science
- Biochemistry
- Natural Products
- Pharmacology
Background
- Taiwanese green propolis is known for its medicinal properties.
- Antibacterial activity is a significant aspect of propolis research.
- Extraction methods are crucial for characterizing natural compounds.
- Ethanol is a common solvent used in extraction protocols.
Purpose of Study
- To establish a protocol for extracting Taiwanese green propolis.
- To characterize the antibacterial properties of the extracted propolis.
- To provide a reliable method for quality assessment.
Methods Used
- Weighing 100 grams of Taiwanese green propolis.
- Grinding the propolis into a fine powder using a spice grinder.
- Preparing five flasks with various concentrations of ethanol in water.
- Following a systematic extraction procedure.
Main Results
- The protocol successfully extracts Taiwanese green propolis.
- Characterization reveals significant antibacterial activity.
- The method is reproducible and reliable for quality assessment.
- Demonstration by Chun'Ting Chen and Yi'Hsuan Chien enhances understanding.
Conclusions
- The extraction protocol is effective for Taiwanese green propolis.
- Antibacterial properties are confirmed through characterization.
- This method can be utilized for further research on propolis.
Frequently Asked Questions
Executive Industry Relevance
Establishing a standardized extraction and analytical protocol for natural products like Taiwanese green propolis supports early-stage target validation by enabling reproducible isolation of bioactive prenylated flavonoids. This approach enhances predictive confidence in lead identification efforts by providing quantifiable, antibacterial activity-linked compound profiles. The method facilitates mechanistic de-risking through consistent quality assessment across seasonal and geographic variability.
Strategic Applications in Biopharma R&D
Early Discovery & Target Validation
- Scientific Value: Enables interrogation of therapeutic hypotheses by isolating and quantifying propolins C, D, F, and G with defined antibacterial activity.
- Operational Value: Provides a repeatable ethanol-based extraction protocol yielding maximal dry matter and propolin concentrations at 95% and 99.5% ethanol concentrations.
- Strategic Value: Supports predictive confidence in prioritizing natural product leads with demonstrated antibacterial effects against S. aureus.
Screening & Assay Development
- Scientific Value: Generates HPLC-quantifiable propolin concentrations correlated with antibacterial activity, enabling compound-specific activity tracking.
- Operational Value: Delivers standardized extracts with reproducible turbidity-based MIC measurements in 96-well plates against S. aureus.
- Strategic Value: Establishes a scalable assay platform for screening natural extracts with defined inhibitory thresholds (10–20 µg/mL MIC range).
Translational & Preclinical Research
- Scientific Value: Supports disease-relevant system modeling by linking propolin concentration to antibacterial outcomes in preclinical infection models.
- Operational Value: Ensures continuity from discovery through standardized extract preparation and storage at –20°C prior to analysis.
- Strategic Value: Informs risk-adjusted advancement decisions by identifying extraction conditions that maximize bioactive yield.
Pipeline & Workflow Integration
This method integrates into the discovery continuum from early biological screening to lead optimization by delivering quantified, bioactive natural product fractions suitable for downstream profiling.
- Discovery Biology: Supports hypothesis testing via isolation of prenylated flavonoids with defined antibacterial activity, enabling pathway and target interrogation.
- Screening: Delivers assay-ready extracts with standardized concentration ranges and reproducible antibacterial readouts for compound evaluation.
- Analytics: Provides HPLC-based quantification and UV detection at 280 nm, generating reliable concentration-response data for lead comparison.
- Translational Research: Connects extraction yield to antibacterial activity, supporting preclinical continuity in infection models.
- Enterprise Reuse: Establishes a reusable solvent extraction and analytical framework applicable to other propolis sources or natural product libraries.
Operational & Enterprise Impact
- Scientific Value: Increases predictive confidence in natural product lead validation through quantified propolin and antibacterial activity correlation.
- Operational Value: Ensures reproducibility via standardized ethanol extraction, filtration, reconstitution, and HPLC analysis steps.
- Strategic Value: Improves go/no-go decisions by identifying optimal ethanol concentrations (95%, 99.5%) for maximal bioactive yield.
- Portfolio Impact: Enables risk-adjusted prioritization of natural product batches based on seasonal and geographic propolin variability.
Implementation Considerations
- Requires expertise in natural product extraction, HPLC operation, and microbiological assay techniques.
- Dependent on access to HPLC systems with UV detection at 280 nm, reverse-phase columns, and temperature-controlled incubators.
- Necessitates cross-team standardization of extract preparation, storage (–20°C), and MIC assay protocols.
- Involves adaptation considerations for varying propolis sources due to seasonal and geographic influences on propolin concentration.
- Practical limitation: Antibacterial activity was not observed against E. coli under tested conditions, indicating compound-specific efficacy.
Biopharma FAQs
Chapters in this video
0:04
Title
0:40
Preparation of Ethanol-extracted Compounds and Preparation of Ethanol Extracts for HPLC
2:22
Analysis of the Propolin Content Using HPLC
3:57
Minimum Inhibitory Concentration and Minimum Bactericidal Concentration Analysis
5:16
Results: Analysis of Extracted Taiwanese Green Propolis
6:30
Conclusion
