This manuscript was edited by Wallace Academic Editing.

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 and shake the flask at 250 rpm for 48 h.</li>
	<li>Filter the ethanol extracts through filter paper with a 25 &#956;m pore size.</li>
	<li>Reconstitute the filtrates to their original volume (100 mL) with 95% ethanol using a volumetric flask.</li>
	<li>Store the ethanol extracts at -20 &#176;C. 
	NOTE: The protocol can be paused here.</li>
</ol>
2. Preparation of Ethanol Extracts for HPLC
<ol>
	<li>Concentrate 10 mL of ethanol extracts by vacuum evaporation at 40 &#176;C for 15 min.</li>
	<li>Bake the dry matter at 45 &#176;C for 24 h.</li>
	<li>Reconstitute the dry matter with 10 mL of 95% ethanol.</li>
	<li>Filter 1 mL of ethanol extracts using a sterile syringe filter with a 0.45 &#181;m pore size.</li>
	<li>Refilter the ethanol extracts using a sterile syringe filter with a 0.22 &#181;m pore size. The filtrate is collected and can be directly analyzed using HPLC.</li>
</ol>
3. Analysis of the Propolin Content Using HPLC
<ol>
	<li>Establishment of standard curves of propolins
	<ol>
		<li>Prepare 1 L of the mobile phase of 88.8:11.2 (v/v) methanol:water solution.</li>
		<li>Prepare serial dilutions of propolin standard (C, D, F, and G) concentrations (15.625 mg/mL, 31.25 mg/mL, 62.5 mg/mL, and 125 mg/mL, respectively) using the mobile phase solution as a solvent.</li>
		<li>Inject 20 &#181;L of propolin standard concentrations into the reverse-phase column, sequentially from the low concentration to the high concentration.</li>
		<li>Set the HPLC column at 30 &#176;C and the flow rate to 1 mL/min.</li>
		<li>Set the wavelength of the UV detector to 280 nm and the recorder time to 20 min.</li>
		<li>Analyze the standards at least 3x.</li>
		<li>Plot the measurement response (y-axis) against the concentration (x-axis) using calculation sheet software, and create a standard curve with the equation and R-square value.</li>
	</ol>
	</li>
	<li>Analysis of ethanol extracts
	<ol>
		<li>Inject 20 &#181;L of the ethanol extracts obtained from step 2.5 into the reverse-phase column.</li>
		<li>Set the HPLC column at 30 &#176;C and the flow rate to 1 mL/min.</li>
		<li>Set the wavelength of the UV detector to 280 nm and the recorder time to 20 min.</li>
		<li>Analyze the standards at least 3x.</li>
		<li>Calculate the concentration of propolin in the ethanol extract, using the equation for the standard curve obtained from step 3.1.7 that uses the peak area for each propolin.</li>
	</ol>
	</li>
</ol>
4. Minimum Inhibitory Concentration and Minimum Bactericidal Concentration Analysis
NOTE: The microdilution method is used to evaluate the antibacterial efficacy of ethanol-extracted propolins from Taiwanese green propolis.
<ol>
	<li>Preparation of test organisms
	<ol>
		<li>Thaw the bacterial strains, Staphylococcus aureus and Escherichia coli, and then, culture them in tryptic soy broth and nutrient broth, respectively, at 37 &#176;C for 24 h.</li>
		<li>Passage the S. aureus using tryptic soy broth and E. coli using nutrient broth for two generations and, then, calculate colony-forming units by counting individual colonies on an agar plate.</li>
	</ol>
	</li>
	<li>Preparation of ethanol extracts for antibacterial activity testing
	<ol>
		<li>Concentrate the ethanol extracts obtained from step 1.5 by vacuum evaporation at 40 &#176;C for 15 min.</li>
		<li>Reconstitute the dry matter with dimethyl sulfoxide (DMSO) and adjust the concentration of the extract to 12.8 mg/mL.</li>
		<li>Make a serial dilution (concentrations: 5, 10, 20, 40, 80, 160, 320, and 640 &#181;g/mL) of ethanol extracts using the broth.</li>
	</ol>
	</li>
	<li>Minimum inhibitory concentration tests
	<ol>
		<li>Add 10 &#181;L of diluted ethanol extracts ranging from 0.156 to 640.0 &#181;g/mL into a 96-well plate.</li>
		<li>Using broth, adjust the volume to 100 &#181;L, and maintain 5% DMSO in all the dilutions.</li>
		<li>Inoculate 100 &#181;L of bacterial culture (1 x 106/mL) into the 96-well plate.</li>
		<li>Culture inoculum containing various concentrations of ethanol extracts at 37 &#176;C for 48 h.</li>
		<li>Analyze the bacterial growth according to turbidity and using optical density (microplate reader) at 590 nm to determine the minimum inhibitory concentration (MIC).</li>
	</ol>
	</li>
	<li>Minimum bactericidal concentration tests
	<ol>
		<li>Inoculate 10 &#181;L of liquid culture from each well of the MIC test that exhibited no growth onto an agar plate.</li>
		<li>Incubate at 37 &#176;C for 24 h.</li>
		<li>Determine the bactericidal activity by identifying the lowest concentration that revealed no visible bacterial growth. The concentration that completely eliminates cell growth is considered to be the minimum bacterial concentration (MBC).</li>
	</ol>
	</li>
</ol>

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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. 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The authors have nothing to declare.

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 propolis19,21,22. It was determined that bioactive compounds in Brazilian propolis could be differentially extracted depending on the percentage of ethanol19. The extraction efficiency of bioactive compounds in propolis may vary because of the different chemical compositions of Brazilian propolis and Taiwanese green propolis. Here, we provide a reliable and repeatable protocol for extracting Taiwanese green propolis. The bioactive compounds in Taiwanese green propolis could be harvested within 2 days, using ethanol as a solvent. A similar finding also supports the protocol presented here and determined that propolins (C, D, F, and G) from Taiwanese green propolis could be extracted using 95% ethanol in combination with a 3 day extraction23. Another study demonstrated that 95% ethanol in combination with a 21 h extraction was able to harvest propolins from Taiwanese green propolis24. However, the exact concentration of propolins after the 21 h ethanol extraction must be verified. Researchers have proposed that the heating process increases the extraction efficiency for Brazilian propolis19,21,22. Whether propolins from Taiwanese green propolis can be extracted by heating must be investigated.
The ethanol extracts from Taiwanese green propolis have antibacterial effects against gram-positive pathogens. Because propolis is hydrophobic, studies have demonstrated that an organic solvent such as ethanol is a suitable solvent for propolis extraction8,16,17,18,19,20. Studies have also demonstrated that an increased ethanol concentration leads to more extracted bioactive compounds8,17,18,20. In the present protocol, the maximum dry matter yields of Taiwanese green propolis are observed in the 95% and 99.5% ethanol extracts, as well as the highest concentration of propolins and antibacterial activity.
The present protocol is designed for Taiwanese green propolis extraction using ethanol, and the quality assessment is made on the basis of the contents of propolins. However, some ethanol-insoluble bioactive compounds in Taiwanese green propolis cannot properly be isolated using the present protocol.
The most crucial aspect of the method with respect to existing methods is that this method saves time relative to methods proposed in other studies17,23. Moreover, the 95% and 99.5% ethanol extracts of Taiwanese green propolis exhibited a high concentration of propolins and antibacterial activity.
This approach has direct applications for characterizing other unknown ethanol-soluble bioactive compounds in Taiwanese green propolis. Additionally, ethanol extracts containing propolins could be used for determining other bioactivities.
One of the key experimental procedures is ensuring the uniformity of the Taiwanese green propolis during grinding (protocol step 1.1). Inappropriate grinding of Taiwanese green propolis can lead to low dry matter yields and propolin content. It is essential to use a proper grinding apparatus, such as a spice grinder, tissue grinder, or homogenizer, and to take care that the propolis becomes a fine powder without any large particles remaining after grinding.

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&amp;Uuml;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, performs best for Taiwanese green propolis extraction. The signal of standard propolins C, D, F, and G was identified and quantified using HPLC (Figure 1a). The signal of propolins in the ethanol extracts was characterized using individual propolin standards and HPLC (Figure 1b). Normally, the concentration of propolins (C, D, F, and G) in Taiwanese green propolis is positively associated with the ethanol concentration during extraction (Table 1). The highest yield of propolins in Taiwanese green propolis was produced in the 95% and 99.5% ethanol extracts.
Positive representative data for the antibacterial effect of ethanol extracts are presented in Table 2. Antibacterial activity against S. aureus and E. coli in the ethanol extracts were examined. The average MIC and MBC of ethanol extracts for S. aureus were 10 - 20 &#181;g/mL and 20 &#181;g/mL, respectively (Table 2). Water extracts did not have antibacterial effects against S. aureus (Table 2). No antibacterial effect on E. coli was observed with either the ethanol or water extracts (Table 2).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/58743/58743fig1.jpg" /> 
Figure 1: Identification of propolins in Taiwanese green propolis. These panels show (a) standards of propolins and (b) the measurement of propolins in Taiwanese green propolis using HPLC. This figure has been modified from Chen et al.8. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58743/58743fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td rowspan="2">Solvent*</td>
			<td rowspan="2">Yield (%)</td>
			<td>Propolin (C+D+F+G)</td>
		</tr>
		<tr>
			<td>(mg/mL)</td>
		</tr>
		<tr>
			<td>99.5% EtOH</td>
			<td>66.75 &#177; 0.5a**</td>
			<td>36.73 &#177; 0.80a</td>
		</tr>
		<tr>
			<td>95% EtOH</td>
			<td>66.25 &#177; 0.50a</td>
			<td>37.55 &#177; 1.29a</td>
		</tr>
		<tr>
			<td>80% EtOH</td>
			<td>64.75 &#177; 0.96b</td>
			<td>34.25 &#177; 0.71b</td>
		</tr>
		<tr>
			<td>70% EtOH</td>
			<td>63.25 &#177; 0.96c</td>
			<td>31.53 &#177; 0.31c</td>
		</tr>
		<tr>
			<td>60% EtOH</td>
			<td>59.00 &#177; 1.41d</td>
			<td>29.34 &#177; 1.59d</td>
		</tr>
		<tr>
			<td>Water</td>
			<td>7.00 &#177; 0.82e</td>
			<td>ND***</td>
		</tr>
	</tbody>
</table>
Table 1: Dry matter yield (%) and propolin content (mg/mL) in Taiwanese green propolis extracted using various solvents. * 10 g of propolis was extracted using 100 mL of solvent, and extracts were finally reconstituted to 100 mL. ** The values are the mean &#177; the standard deviation (SD). a-e Means within a column that have no common superscript are significantly different (P &#60; 0.05). *** ND = not detected. This table has been modified from Chen et al.8.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td rowspan="2">Extract</td>
			<td colspan="2">Staphylococcus aureus</td>
			<td>Escherichia coli</td>
		</tr>
		<tr>
			<td>MIC (&#181;g/mL)</td>
			<td>MBC (&#181;g/mL)</td>
			<td>MIC (&#181;g/mL)</td>
		</tr>
		<tr>
			<td>99.5% EtOH</td>
			<td>10</td>
			<td>20</td>
			<td>&#62;640</td>
		</tr>
		<tr>
			<td>95% EtOH</td>
			<td>10</td>
			<td>20</td>
			<td>&#62;640</td>
		</tr>
		<tr>
			<td>80% EtOH</td>
			<td>20</td>
			<td>20</td>
			<td>&#62;640</td>
		</tr>
		<tr>
			<td>70% EtOH</td>
			<td>10</td>
			<td>20</td>
			<td>&#62;640</td>
		</tr>
		<tr>
			<td>60% EtOH</td>
			<td>20</td>
			<td>20</td>
			<td>&#62;640</td>
		</tr>
		<tr>
			<td>Water</td>
			<td>ND</td>
			<td>ND</td>
			<td>ND</td>
		</tr>
	</tbody>
</table>
Table 2: MIC and MBC (&#181;g/mL) of various extracts against S. aureus and E. coli. This table has been modified from Chen et al.8.

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 ...

extraction-and-analysis-of-taiwanese-green-propolis

Nanyang Technological University

Research

JoVE Journal

Biochemistry

8.4K Views.  National Ilan University. Hier presenteren we het protocol voor het gebruik van ethanol als oplosmiddel om Taiwanese groene propolis te extraheren en te karakteriseren die antibacteriële activiteit remt. Dit protocol is een betrouwbare en herhaalbare methode voor het bepalen van de kwaliteit van taiwanese groene propolis. Demonstrerende procedure zijn Chun'Ting Chen en Yi'Hsuan Chien, promovendi van mijn laboratorium.Om te beginnen, wegen 100 gram Taiwanese groene propolis. Maal met behulp van een kruidenmolen...

Video: Extraction and Analysis of Taiwanese Green Propolis

Transcript and Metabolite Profiling for the Evaluation of Tobacco Tree and Poplar as Feedstock for the Bio-based Industry

The global demand for food, feed, energy, and water poses extraordinary challenges for future generations. It is evident that robust platforms for the exploration of renewable resources are necessary to overcome these challenges. Within the multinational framework MultiBioPro we are developing biorefinery pipelines to maximize the use of plant biomass. More specifically, we use poplar and tobacco tree (Nicotiana glauca) as target crop species for improving saccharification, isoprenoid, long chain hydrocarbon contents, fiber quality, and suberin and lignin contents. The methods used to obtain these outputs include GC-MS, LC-MS and RNA sequencing platforms. The metabolite pipelines are well established tools to generate these types of data, but also have the limitations in that only well characterized metabolites can be used. The deep sequencing will allow us to include all transcripts present during the developmental stages of the tobacco tree leaf, but has to be mapped back to the sequence of Nicotiana tabacum. With these set-ups, we aim at a basic understanding for underlying processes and at establishing an industrial framework to exploit the outcomes. In a more long term perspective, we believe that data generated here will provide means for a sustainable biorefinery process using poplar and tobacco tree as raw material. To date the basal level of metabolites in the samples have been analyzed and the protocols utilized are provided in this article.

Plant biomass offers a renewable resource for multiple products, including fuel, feed, food, and a variety of materials. In this paper we investigate the properties of tobacco tree (Nicotiana glauca) and poplar as suitable sources for a biorefinery pipeline.

The global demand for food, feed, energy, and water poses extraordinary challenges for future generations. It is evident that robust platforms for the ...

Environment

Empirical, Metagenomic, and Computational Techniques Illuminate the Mechanisms by which Fungicides Compromise Bee Health

Growers often use fungicide sprays during bloom to protect crops against disease, which exposes bees to fungicide residues. Although considered &#34;bee-safe,&#34; there is mounting evidence that fungicide residues in pollen are associated with bee declines (for both honey and bumble bee species). While the mechanisms remain relatively unknown, researchers have speculated that bee-microbe symbioses are involved. Microbes play a pivotal role in the preservation and/or processing of pollen, which serves as nutrition for larval bees. By altering the microbial community, it is likely that fungicides disrupt these microbe-mediated services, and thereby compromise bee health. This manuscript describes the protocols used to investigate the indirect mechanism(s) by which fungicides may be causing colony decline. Cage experiments exposing bees to fungicide-treated flowers have already provided the first evidence that fungicides cause profound colony losses in a native bumble bee (Bombus impatiens). Using field-relevant doses of fungicides, a series of experiments have been developed to provide a finer description of microbial community dynamics of fungicide-exposed pollen. Shifts in the structural composition of fungal and bacterial assemblages within the pollen microbiome are investigated by next-generation sequencing and metagenomic analysis. Experiments developed herein have been designed to provide a mechanistic understanding of how fungicides affect the microbiome of pollen-provisions. Ultimately, these findings should shed light on the indirect pathway through which fungicides may be causing colony declines.

Microbial consortia within bumble bee hives enrich and preserve pollen for bee larvae. Using next generation sequencing, along with laboratory and field-based experiments, this manuscript describes protocols used to test the hypothesis that fungicide residues alter the pollen microbiome, and colony demographics, ultimately leading to colony loss.

Growers often use fungicide sprays during bloom to protect crops against disease, which exposes bees to fungicide residues. Although considered ...

Transient Expression in Nicotiana Benthamiana Leaves for Triterpene Production at a Preparative Scale

The triterpenes are one of the largest and most structurally diverse families of plant natural products. Many triterpene derivatives have been shown to possess medicinally relevant biological activity. However, thus far this potential has not translated into a plethora of triterpene-derived drugs in the clinic. This is arguably (at least partially) a consequence of limited practical synthetic access to this class of compound, a problem that can stifle the exploration of structure-activity relationships and development of lead candidates by traditional medicinal chemistry workflows. Despite their immense diversity, triterpenes are all derived from a single linear precursor, 2,3-oxidosqualene. Transient heterologous expression of biosynthetic enzymes in N. benthamiana can divert endogenous supplies of 2,3-oxidosqualene towards the production of new high-value triterpene products that are not naturally produced by this host. Agro-infiltration is an efficient and simple means of achieving transient expression in N. benthamiana. The process involves infiltration of plant leaves with a suspension of Agrobacterium tumefaciens carrying the expression construct(s) of interest. Co-infiltration of an additional A. tumefaciens strain carrying an expression construct encoding an enzyme that boosts precursor supply significantly increases yields. After a period of five days, the infiltrated leaf material can be harvested and processed to extract and isolate the resulting triterpene product(s). This is a process that is linearly and reliably scalable, simply by increasing the number of plants used in the experiment. Herein is described a protocol for rapid preparative-scale production of triterpenes utilizing this plant-based platform. The protocol utilizes an easily replicable vacuum infiltration apparatus, which allows the simultaneous infiltration of up to four plants, enabling batch-wise infiltration of hundreds of plants in a short period of time.

Transient Expression in Nicotiana Benthamiana Leaves for Triterpene Production at a Preparative Scale

Transient heterologous expression of biosynthetic enzymes in Nicotiana benthamiana leaves can divert endogenous supplies of 2,3-oxidosqualene towards the production of new high-value triterpene products. Herein is described a detailed protocol for rapid (5 days) preparative-scale production of triterpenes and analogs utilizing this powerful plant-based platform.

The triterpenes are one of the largest and most structurally diverse families of plant natural products. Many triterpene derivatives have been shown to ...

Fizzy Extraction of Volatile Organic Compounds Combined with Atmospheric Pressure Chemical Ionization Quadrupole Mass Spectrometry

Chemical analysis of volatile and semivolatile compounds dissolved in liquid samples can be challenging. The dissolved components need to be brought to the gas phase, and efficiently transferred to a detection system. Fizzy extraction takes advantage of the effervescence phenomenon. First, a carrier gas (here, carbon dioxide) is dissolved in the sample by applying overpressure and stirring the sample. Second, the sample chamber is decompressed abruptly. Decompression leads to the formation of numerous carrier gas bubbles in the sample liquid. These bubbles assist the release of the dissolved analyte species from the liquid to the gas phase. The released analytes are immediately transferred to the atmospheric pressure chemical ionization interface of a triple quadrupole mass spectrometer. The ionizable analyte species give rise to mass spectrometric signals in the time domain. Because the release of the analyte species occurs over short periods of time (a few seconds), the temporal signals have high amplitudes and high signal-to-noise ratios. The amplitudes and areas of the temporal peaks can then be correlated with concentrations of the analytes in the liquid samples subjected to fizzy extraction, which enables quantitative analysis. The advantages of fizzy extraction include: simplicity, speed, and limited use of chemicals (solvents).

Fizzy extraction is a new laboratory technique for analysis of volatile and semivolatile compounds. A carrier gas is dissolved in the liquid sample by applying overpressure and stirring the sample. The sample chamber is then decompressed. The analyte species are liberated to the gas phase due to effervescence.

Chemical analysis of volatile and semivolatile compounds dissolved in liquid samples can be challenging. The dissolved components need to be brought to ...

Chemistry

A Customizable Approach for the Enzymatic Production and Purification of Diterpenoid Natural Products

Diterpenoids form a diverse class of small molecule natural products that are widely distributed across the kingdoms of life and have critical biological functions in developmental processes, interorganismal interactions, and environmental adaptation. Due to these various bioactivities, many diterpenoids are also of economic importance as pharmaceuticals, food additives, biofuels, and other bioproducts. Advanced genomics and biochemical approaches have enabled a rapid increase in the knowledge of diterpenoid-metabolic genes, enzymes, and pathways. However, the structural complexity of diterpenoids and the narrow taxonomic distribution of individual compounds in often only a single species remain constraining factors for their efficient production. Availability of a broader range of metabolic enzymes now provide resources for producing diterpenoids in sufficient titers and purity to facilitate a deeper investigation of this important metabolite group. Drawing on established tools for microbial and plant-based enzyme co-expression, we present an easily operated and customizable protocol for the enzymatic production of diterpenoids in either Escherichia coli or Nicotiana benthamiana, and the purification of desired products via silica chromatography and semi-preparative HPLC. Using the group of maize (Zea mays) dolabralexin diterpenoids as an example, we highlight how modular combinations of diterpene synthase (diTPS) and cytochrome P450 monooxygenase (P450) enzymes can be used to generate different diterpenoid scaffolds. Purified compounds can be used in various downstream applications, such as metabolite structural analyses, enzyme structure-function studies, and in vitro and in planta bioactivity experiments.

Here we present easy to use protocols for producing and purifying diterpenoid metabolites through the combinatorial expression of biosynthetic enzymes in Escherichia coli or Nicotiana benthamiana, followed by chromatographic product purification. The resulting metabolites are suitable for various studies including molecular structure characterization, enzyme functional studies, and bioactivity assays.

Diterpenoids form a diverse class of small molecule natural products that are widely distributed across the kingdoms of life and have critical biological ...

Source and Route of Pyrrolizidine Alkaloid Contamination in Tea Samples

Toxic pyrrolizidine alkaloids (PAs) are found in tea samples, which pose a threat to human health. However, the source and route of PA contamination in tea samples have remained unclear. In this work, an adsorbent method combined with UPLC-MS/MS was developed to determine 15 PAs in the weed Ageratum conyzoides L., A. conyzoides rhizospheric soil, fresh tea leaves, and dried tea samples. The average recoveries ranged from 78%-111%, with relative standard deviations of 0.33%-14.8%. Fifteen pairs of A. conyzoides and A. conyzoides rhizospheric soil&#160;samples and 60 fresh tea leaf samples were collected from the Jinzhai tea garden in Anhui Province, China, and analyzed for the 15 PAs. Not all 15 PAs were detected in fresh tea leaves, except for intermedine-N-oxide (ImNO) and senecionine (Sn). The content of ImNO (34.7 &#181;g/kg) was greater than that of Sn (9.69 &#181;g/kg). In addition, both ImNO and Sn were concentrated in the young leaves of the tea plant, while their content was lower in the old leaves. The results indicated that the PAs in tea were transferred through the path of PA-producing weeds-soil-fresh tea leaves in tea gardens.

The present protocol describes the contamination of pyrrolizidine alkaloids (PAs) in tea samples from PA-producing weeds in tea gardens.

Toxic pyrrolizidine alkaloids (PAs) are found in tea samples, which pose a threat to human health. However, the source and route of PA contamination in ...

Extraction of Lignin with High &#946;-O-4 Content by Mild Ethanol Extraction and Its Effect on the Depolymerization Yield

Lignin valorization strategies are a key factor for achieving more economically competitive biorefineries based on lignocellulosic biomass. Most of the emerging elegant procedures to obtain specific aromatic products rely on the lignin substrate having a high content of the readily cleavable &#946;-O-4 linkage as present in the native lignin structure. This provides a miss-match with typical technical lignins that are highly degraded and therefore are low in &#946;-O-4 linkages. Therefore, the extraction yields, and the quality of the obtained lignin are of utmost importance to access new lignin valorization pathways. In this manuscript, a simple protocol is presented to obtain lignins with high &#946;-O-4 content by relatively mild ethanol extraction that can be applied to different lignocellulose sources. Furthermore, analysis procedures to determine the quality of the lignins are presented together with a depolymerization protocol that yields specific phenolic 2-arylmethyl-1,3-dioxolanes, which can be used to evaluate the obtained lignins. The presented results demonstrate the link between lignin quality and potential for the lignins to be depolymerized into specific monomeric aromatic chemicals. Overall, the extraction and depolymerization demonstrates a trade-off between the lignin extraction yield and the retention of the native aryl-ether structure and thus the potential of the lignin to be used as the substrate for the production of chemicals for higher-value applications.

Here, we present a protocol to perform ethanol extraction of lignin from several biomass sources. The effect of the extraction conditions on the lignin yield and &#946;-O-4 content are presented. Selective depolymerization is performed on the obtained lignins to obtain high aromatic monomer products.

Lignin valorization strategies are a key factor for achieving more economically competitive biorefineries based on lignocellulosic biomass. Most of the ...

DNA Barcoding for Medicinal Plants: Identification and Phylogenetic Analysis

Application of DNA Barcoding to Identify Medicinal Plants

Medicinal plants are valuable resources globally and are used worldwide to maintain health and treat disease; however, the presence of adulteration obstructs their development. DNA barcoding, a technique for species identification by standard DNA regions, facilitates prompt and accurate identification of traditional medicinal plants. The process of DNA barcoding entails six basic steps: 1) processing the medicinal plants, 2) extracting high-quality total DNA from the medicinal plants using centrifugal column method, 3) amplifying target DNA region internal transcribed spacer 2 (ITS2) with universal primers of plants and performing Sanger sequencing, 4) splicing and aligning sequence to obtain the target sequence, 5) matching the barcode sequence against the barcode library for identification, 6) aligning sequence, comparing intraspecific and interspecific variation, constructing phylogenetic neighbor-joining tree. As shown in the results, the universal primer can amplify the target region. Basic Local Alignment Search Tool (BLAST) demonstrates the percentage identified was 100%, and the neighbor-joining tree demonstrates that the splicing sequences were clustered with the A. sinensis OR879715.1 clade, and the clade support value is 100. This protocol provides a reference for applying DNA barcoding technology as an effective method to identify medicinal plants and adulterants.

Author Spotlight: Harnessing DNA Barcode Technology to Enhance the Efficiency of Medicinal Plant Identification

The present protocol describes the use of DNA barcoding technology to authenticate plant-based medicinal materials, and the medicinal plant Angelica sinensis (Oliv.) Diels was identified as an example.

Medicinal plants are valuable resources globally and are used worldwide to maintain health and treat disease; however, the presence of adulteration ...

Genetics

Unveiling Tea Aroma Complexity with Solvent-Assisted Flavor Evaporation Method

Tea Aroma Analysis Based on Solvent-Assisted Flavor Evaporation Enrichment

Tea aroma is an important factor in tea quality, but it is challenging to analyze due to the complexity, low concentration, diversity, and lability of the volatile components of tea extract. This study presents a method for obtaining and analyzing the volatile components of tea extract with odor preservation using solvent-assisted flavor evaporation (SAFE) and solvent extraction followed by gas chromatography-mass spectrometry (GC-MS). SAFE is a high-vacuum distillation technique that can isolate volatile compounds from complex food matrices without any non-volatile interference. A complete step-by-step procedure for tea aroma analysis is presented in this article, including the tea infusion preparation, solvent extraction, SAFE distillation, extract concentration, and analysis by GC-MS. This procedure was applied to two tea samples (green tea and black tea), and qualitative as well as quantitative results on the volatile composition of the tea samples were obtained. This method can not only be used for the aroma analysis of various types of tea samples but also for molecular sensory studies on them.

Author Spotlight: Exploring Tea Aroma Using Solvent-Assisted Flavor Evaporation Technique 

Presented here is a method for enriching and analyzing the volatile components of tea extracts using solvent-assisted flavor evaporation and solvent extraction followed by gas chromatography-mass spectrometry, which can be applied to all types of tea samples.

Tea aroma is an important factor in tea quality, but it is challenging to analyze due to the complexity, low concentration, diversity, and lability of the ...

Ultra High-Performance Liquid Chromatography-Mass Spectrometry and Self-established Database Analysis of Chinese Herbal Medicine Components

Chinese herbal medicine is complex and has numerous unknown compounds, making qualitative research crucial. Ultra-high-performance liquid chromatography quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF-MS) is the most widely used method in qualitative analysis of compounds. The method includes standardized and programmed protocols for sample pretreatment, MS tune, MS acquisition, and data processing. The sample pretreatments include collection, pulverization, solvent extraction, ultrasound, centrifugation, and filtration. Data post-processing was described in detail and includes data importing, self-established database construction, method establishment, data processing, and other manual operations. The above-ground part of the alpine yarrow herb, Achillea millefolium L., is used to treat inflammation, gastrointestinal disturbances, and pain and its 3-oxa-guaianolides could be useful leads for anti-inflammatory drug development. Three representative compounds in AML were identified, combining TOF-MS with a self-established database. Moreover, the differences from existing literature, liquid-phase parameter optimization, scan mode selection, ion source suitability, collision energy adjustment, isomer screening, method limitation, and possible solutions were discussed. This standardized analysis method is universal and can be applied to identify complex compounds in Chinese herbal medicine.

We describe a general protocol and systematic design that could be applied to separate and recognize complex components in alpine yarrow herb, Achillea millefolium L., a Chinese herbal medicine.

Chinese herbal medicine is complex and has numerous unknown compounds, making qualitative research crucial. Ultra-high-performance liquid chromatography ...

Extraction and Analysis of Taiwanese Green Propolis

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