Name: extraction and analysis of taiwanese green propolis
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1. Preparation of Ethanol-extracted Compounds
<ol>
	<li>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.</li>
	<li>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.</li>
	<li>Incubate at 25 &#176;C...

<ol>
	<li>Zabaiou, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biological+properties+of+propolis+extracts:+Something+new+from+an+ancient+product.">Biological properties of propolis extracts: Something new from an ancient product.</a> Chemistry and Physics of Lipids. 207 (Pt B), 214-222 (2017).</li><li>Bankova, V. B., De Castro, S. L., Marcucci, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Propolis:+Recent+advances+in+chemistry+and+plant+origin.">Propolis: Recent advances in chemistry and plant origin.</a> Apidologie. 31 (1), 3-15 (2000).</li><li>Park, Y. K., Alencar, S. M., Aguiar, C. 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K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+radical+scavenging+activity,+cytotoxic+effects+and+apoptosis+induction+in+human+melanoma+cells+by+Taiwanese+propolis+from+different+sources.">Comparison of radical scavenging activity, cytotoxic effects and apoptosis induction in human melanoma cells by Taiwanese propolis from different sources.</a> Evidence-Based Complementary and Alternative. 1 (2), 175-185 (2004).</li><li>Chen, C. N., Wu, C. L., Lin, J. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Apoptosis+of+human+melanoma+cells+induced+by+the+novel+compounds+propolin+A+and+propolin+B+from+Taiwanese+propolis.">Apoptosis of human melanoma cells induced by the novel compounds propolin A and propolin B from Taiwanese propolis.</a> Cancer Letters. 24 (1-2), 218-231 (2007).</li><li>Huang, W. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Propolin+G,+a+prenylflavanone,+isolated+from+Taiwanese+propolis,+induces+caspase-dependent+apoptosis+in+brain+cancer+cells.">Propolin G, a prenylflavanone, isolated from Taiwanese propolis, induces caspase-dependent apoptosis in brain cancer cells.</a> Journal of Agricultural and Food Chemistry. 55 (18), 7366-7376 (2007).</li><li>Chen, Y. W., Ye, S. R., Ting, C., Yu, Y. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antibacterial+activity+of+propolins+from+Taiwanese+green+propolis.">Antibacterial activity of propolins from Taiwanese green propolis.</a> Journal of Food and Drug Analysis. 26 (2), 761-768 (2018).</li><li>Hegazi, A. G., Abd El Hady, F. K., Abd Allah, F. A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemical+composition+and+antimicrobial+activity+of+European+propolis.">Chemical composition and antimicrobial activity of European propolis.</a> Zeitschrift f&#252;r Naturforschung. 55 (1-2), 70-75 (2000).</li><li>Sforcin, J. M., Fernandes, A., Lopes, C. A. M., Bankova, V., Funari, S. R. 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W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antibacterial+properties+of+propolis+(bee+glue).">Antibacterial properties of propolis (bee glue).</a> Journal of the Royal Society of Medicine. 83 (3), 159-160 (1990).</li><li>Kujumgiev, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antibacterial,+antifungal+and+antiviral+activity+of+propolis+from+different+geographic+origins.">Antibacterial, antifungal and antiviral activity of propolis from different geographic origins.</a> Journal of Ethnopharmacology. 64 (3), 235-240 (1999).</li><li>Krol, W., Scheller, S., Shani, J., Pietsz, G., Czuba, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synergistic+effect+of+ethanol+extract+of+propolis+and+antibiotics+in+the+growth+of+Staphylococcus+aureus.">Synergistic effect of ethanol extract of propolis and antibiotics in the growth of Staphylococcus aureus.</a> Drug Research. 43 (5), 607-609 (1993).</li><li>Lu, L. C., Chen, Y. W., Chou, C. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antibacterial+and+DPPH+free+radical-scavenging+activities+of+the+ethanol+extract+of+propolis+collected+in+Taiwan.">Antibacterial and DPPH free radical-scavenging activities of the ethanol extract of propolis collected in Taiwan.</a> Journal of Food and Drug Analysis. 11 (4), 277-282 (2003).</li><li>Tosi, B., Donini, A., Romagnoli, C., Bruni, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antimicrobial+activity+of+some+commercial+extracts+of+propolis+prepared+with+different+solvents.">Antimicrobial activity of some commercial extracts of propolis prepared with different solvents.</a> Phytotherapy Research. 10 (4), 335-336 (1996).</li><li>Cunha, I. B. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Factors+that+influence+the+yield+and+composition+of+Brazilian+propolis+extracts.">Factors that influence the yield and composition of Brazilian propolis extracts.</a> Journal of the Brazilian Chemical Society. 15 (6), 964-970 (2004).</li><li>Woo, S. O., Hong, I. P., Han, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extraction+properties+of+propolis+with+ethanol+concentration.">Extraction properties of propolis with ethanol concentration.</a> Journal of Apicultural Research. 30 (3), 211-216 (2015).</li><li>Park, Y. K., Ikegaki, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+water+and+ethanolic+extracts+of+propolis+and+evaluation+of+the+preparations.">Preparation of water and ethanolic extracts of propolis and evaluation of the preparations.</a> Bioscience, Biotechnology, and Biochemistry. 62 (11), 2230-2232 (1998).</li><li>Ramanauskien&#279;, K., Ink&#279;nien&#279;, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Propolis+oil+extract:+quality+analysis+and+evaluation+of+its+antimicrobial+activity.">Propolis oil extract: quality analysis and evaluation of its antimicrobial activity.</a> Natural Product Research. 25 (15), 1463-1468 (2011).</li><li>Machado, B. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemical+composition+and+biological+activity+of+extracts+obtained+by+supercritical+extraction+and+ethanolic+extraction+of+brown,+green+and+red+propolis+derived+from+different+geographic+regions+in+brazil.">Chemical composition and biological activity of extracts obtained by supercritical extraction and ethanolic extraction of brown, green and red propolis derived from different geographic regions in brazil.</a> PLoS One. 11 (1), e0145954 (2016).</li><li>Monroy, Y. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Brazilian+green+propolis+extracts+obtained+by+conventional+processes+and+by+processes+at+high+pressure+with+supercritical+carbon+dioxide,+ethanol+and+water.">Brazilian green propolis extracts obtained by conventional processes and by processes at high pressure with supercritical carbon dioxide, ethanol and water.</a> The Journal of Supercritical Fluids. 130, 189-197 (2017).</li><li>Popova, M., Chen, C. N., Chen, P. Y., Huang, C. Y., Bankova, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+validated+spectrophotometric+method+for+quantification+of+prenylated+flavanones+in+pacific+propolis+from+Taiwan.">A validated spectrophotometric method for quantification of prenylated flavanones in pacific propolis from Taiwan.</a> Phytochemical Analysis. 21 (2), 186-191 (2010).</li><li>Chen, L. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Taiwanese+green+propolis+ethanol+extract+delays+the+progression+of+type+2+diabetes+mellitus+in+rats+treated+with+streptozotocin/high-fat+diet.">Taiwanese green propolis ethanol extract delays the progression of type 2 diabetes mellitus in rats treated with streptozotocin/high-fat diet.</a> Nutrients. 10 (4), e503 (2018).</li></ol>

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 &#176;C, 50 &#176;C, or 70 &#176;C for 30 min has been proposed for extracting Brazilian propolis<sup c...

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 acids2,3. To date, 10 prenylated flavanone derivatives from Taiwanese green propolis have been identified through high-performance liquid chromatography (HPLC)4,5,6,7. The most abundant among these are propolins C, D, F, and G5,7. The considerable biological effects of Taiwanese green propolis are correlated with its high content of propolins8.
The concentrations of bioactive compounds in propolis vary greatly depending on the season and geographic location from which the propolis is obtained. European propolis mainly contains the flavonoid aglycone and phenolic acids9. The major bioactive compounds in propolis from Brazil are prenylated p-coumaric acids, such as artepillin C10. A study demonstrated that the season is a critical factor for determining the total propolin content in Taiwanese green propolis11. The propolin content in Taiwanese green propolis is highest in summer (May - July) and lowest in winter11. The antibacterial property of propolis has been widely considered to be an indicator of biological activity. Generally, the samples of propolis collected from various regions have exhibited a similar antibacterial property; for example, it is generally effective against almost all gram-positive bacteria and exhibits a limited antibacterial effect against gram-negative bacteria10,12,13. Synergistic interactions between the flavonoids in propolis were demonstrated to have an antibacterial effect14. Similarly, Taiwanese green propolis was reported to have an antimicrobial effect against gram-positive bacteria15. Furthermore, a study also identified an antimicrobial effect from the interactions of propolins in Taiwanese green propolis8.
The characterization of the bioactive compounds in propolis is difficult because its chemical composition can vary according to its source of origin. Therefore, it is necessary to establish a feasible and repeatable method for determining the quality of the Taiwanese green propolis. However, no standard procedure has been established for Taiwanese green propolis extraction and subsequent functional analysis. Several methods that variously apply organic and inorganic solvents have been proposed for propolis extraction16,17,18,19,20. Because propolis is a lipophilic mixture, studies have demonstrated that organic extraction is better than inorganic extraction8,18,19. The total propolin concentration in Taiwanese green propolis and its antibacterial properties are key indicators of the quality of Taiwanese green propolis. Thus, the purpose of this study is to present a protocol for using ethanol as a solvent to extract and characterize the antibacterial properties of Taiwanese green propolis.

<table>
	<tbody>
		<tr>
			<td>ethanol</td>
			<td>Sigma-Aldrich</td>
			<td>E7023</td>
			<td></td>
		</tr>
		<tr>
			<td>Whatman no. 4 filter paper</td>
			<td>Sigma-Aldrich</td>
			<td>WHA1004125</td>
			<td></td>
		</tr>
		<tr>
			<td>methanol</td>
			<td>Sigma-Aldrich</td>
			<td>34860</td>
			<td></td>
		</tr>
		<tr>
			<td>reverse phase RP-18 column</td>
			<td>Phenomenex Inc.</td>
			<td>00G-0234-E0</td>
			<td></td>
		</tr>
		<tr>
			<td>Staphylococcus aureus</td>
			<td>ATCC</td>
			<td>BCRC 10780</td>
			<td></td>
		</tr>
		<tr>
			<td>Escherichia coli</td>
			<td>ATCC</td>
			<td>BCRC 10675</td>
			<td></td>
		</tr>
		<tr>
			<td>tryptic soy broth</td>
			<td>Sigma-Aldrich</td>
			<td>22092</td>
			<td></td>
		</tr>
		<tr>
			<td>nutrient broth</td>
			<td>Sigma-Aldrich</td>
			<td>70122</td>
			<td></td>
		</tr>
		<tr>
			<td>dimethyl sulfoxide</td>
			<td>Sigma-Aldrich</td>
			<td>D2650</td>
			<td></td>
		</tr>
		<tr>
			<td>spice grinder</td>
			<td>Waring</td>
			<td>WSG60K</td>
			<td></td>
		</tr>
		<tr>
			<td>microplate Reader</td>
			<td>Molecular Devices</td>
			<td>EMAX PLUS</td>
			<td></td>
		</tr>
		<tr>
			<td>HPLC system</td>
			<td>Agilent</td>
			<td>1200 Series</td>
			<td></td>
		</tr>
		<tr>
			<td>vacuum evaporation</td>
			<td>B&#220;CHI</td>
			<td>Rotavapor R-215</td>
			<td></td>
		</tr>
	</tbody>
</table>

extraction and analysis of taiwanese green propolis

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.

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...

Watch this Scientific Journal Video about Extraction and Analysis of Taiwanese Green Propolis at JoVE.com

Extraction and Analysis of Taiwanese Green Propolis

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

Taiwanese green propolis is rich in prenylated flavonoids and exhibits a broad range of biological activities, such as antioxidant, antibacterial, and ...

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