Extraction and Analysis of Taiwanese Green Propolis

Summary

We present a protocol for using ethanol as a solvent to extract and characterize Taiwanese green propolis that exhibits antibacterial activity.

Abstract

Taiwanese green propolis is rich in prenylated flavonoids and exhibits a broad range of biological activities, such as antioxidant, antibacterial, and anticancer ones. The bioactive compounds of Taiwanese green propolis are propolins, namely C, D, F, and G. The concentration of propolins in Taiwanese green propolis varies depending on the season and geographic location. Thus, it is critical to establish a standard and repeatable procedure for determining the quality of Taiwanese green propolis. Here, we present a protocol that uses ethanol-based extraction, high-performance liquid chromatography, and an antibacterial activity analysis to characterize Taiwanese green propolis quality. This method indicates that 95% and 99.5% ethanol extractions achieve the maximum dry matter yields from Taiwanese green propolis, thereby yielding the highest concentrations of propolins that have antibacterial properties. According to these findings, the present protocol is deemed reliable and repeatable for determining the quality of Taiwanese green propolis.

Introduction

Propolis is a natural resinous mixture produced by the bee species Apis mellifera. Propolis has been widely used since ancient times in folk medicines. A study recently reported that propolis is beneficial for preventing microbial infections and inflammation¹. Numerous studies have demonstrated that the main bioactive compounds in propolis are flavonoids, phenolic acid esters, prenylated p-coumaric acids, and diterpenic acids^2,3. To date, 10 prenylated flavanone derivatives from Taiwanese green propolis have been identified through high-performance liquid chromatograph....

Protocol

1. Preparation of Ethanol-extracted Compounds

1. Weigh 10 g of frozen Taiwanese green propolis, which was collected from beehives in Taiwan from May to July, and grind it using the spice grinder. Confirm that whole pieces of Taiwanese green propolis are ground into a fine powder without any large particles.
2. Add 100 mL of various concentrations of ethanol (60%, 70%, 80%, 95%, and 99.5%) and water to separate flasks and mix each concentration with 10 g of ground propolis.
3. Incubate at 25 °C and shake the flask at 250 rpm for 48 h.
4. Filter the ethanol extracts through filter paper with a 25 μm pore size.
5. Reconstitu....

Results

Positive representative data for the ethanol extraction are presented in Table 1. The dry matter yield from Taiwanese green propolis was positively associated with the concentration of ethanol. The 95% and 99.5% ethanol extracts had the highest dry matter yield from Taiwanese green propolis. The lowest dry matter yield from Taiwanese green propolis occurred when water was used as the extraction solvent. These results indicate that an organic solvent, such as ethanol, perf.......

Discussion

A study reported that maceration, using various concentrations of ethanol, could be used for Brazilian propolis extraction¹⁷; however, the process was time-consuming¹⁷. It took at least 10 days to extract the bioactive compounds, such as phenolic content, from Brazilian propolis¹⁷. Alternatively, ethanol extraction in combination with heating at 37 °C, 50 °C, or 70 °C for 30 min has been proposed for extracting Brazilian propolis

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
ethanol	Sigma-Aldrich	E7023
Whatman no. 4 filter paper	Sigma-Aldrich	WHA1004125
methanol	Sigma-Aldrich	34860
reverse phase RP-18 column	Phenomenex Inc.	00G-0234-E0
Staphylococcus aureus	ATCC	BCRC 10780
Escherichia coli	ATCC	BCRC 10675
tryptic soy broth	Sigma-Aldrich	22092
nutrient broth	Sigma-Aldrich	70122
dimethyl sulfoxide	Sigma-Aldrich	D2650
spice grinder	Waring	WSG60K
microplate Reader	Molecular Devices	EMAX PLUS
HPLC system	Agilent	1200 Series
vacuum evaporation	BÜCHI	Rotavapor R-215