This study was funded by Mae Fah Luang University. The authors would like to acknowledge the Tea and Coffee Institute of Mae Fah Luang University for facilitating the connection between the researchers and local farmers concerning the acquisition of coffee silverskin samples.

The details of the reagents and the equipment used in this study are listed in the Table of Materials.
1. Experimental preparation
<ol>
	<li>Plant sample preparation
	<ol>
		<li>Collect fresh coffee silverskin (Coffea arabica) and dry it at 60 &#176;C in a tray dryer for 72 h11.</li>
		<li>Grind the dried coffee silverskin (CS) into a fine powder using a grinder and store it at room temperature for further analysis11. 
		NOTE: In this study, fresh CS (C. arabica) was collected from Baan Doi Chang, Mae Suai District, Chiang Rai, Thailand. CS is a byproduct obtained during the hulling process that removes the parchment layer from dried coffee beans after the cherries have been processed to remove the outer fruit layers16.</li>
	</ol>
	</li>
	<li>Chemicals
	<ol>
		<li>Use chemical reagents of analytical grade quality, except for the solvents in the experiment. 
		NOTE: Cosmetic-grade solvents were used in the experiment.</li>
		<li>Use the solvents (water, ethanol, glycerin, propylene glycol, butylene glycol, hexylene glycol, isopentyldiol, 1-2 hexanediol, pentylene glycol, and methylpropanediol) for the extraction of CS with MAE.</li>
	</ol>
	</li>
</ol>
2. Extraction process
<ol>
	<li>Sample and solvent preparation
	<ol>
		<li>Prepare the sample and solvents for the MAE procedure according to a previously reported protocol9 with some modifications.</li>
		<li>Prepare each solvent at 60% concentration by diluting 60 mL of each solvent with distilled water and adjusting the volume to 100 mL for triplicate extractions. 
		NOTE: Use 100% distilled water for water extraction.</li>
		<li>Weigh 0.67 g of CS and mix with 20 mL of each extraction solvent at a 1:30 ratio in a reaction container (Figure 1A) for MAE. 
		NOTE: The maximum solid-liquid amount for each vessel is 2 g of sample and 20 mL of solvent.</li>
		<li>Add a magnetic stirrer bar to each vessel to ensure uniform distribution of the heat and solvent within the sample, enhancing the efficiency of the extraction process and promoting better extraction yield. 
		NOTE: If nonpolar solvents are applied to the extraction process, Teflon stirrer bars can be used instead of magnetic stirrer bars to deliver effective heating through the microwave system.</li>
		<li>Close each vessel firmly with a special tool (Figure 2) and place all vessels into the MAE chamber (Figure 1B).</li>
	</ol>
	</li>
	<li>Setting up the microwave-assisted extraction instrument and procedure
	<ol>
		<li>Perform the extraction procedure according to the reference protocol with some modifications9.</li>
		<li>Open the monitor screen to set up the method by clicking on the toolbox icon on the top bar and selecting the SK eT rotor in the accessory section (Figure 3A,B).</li>
		<li>Select the stirring rate of the stirrer bars by clicking on the stirrer section and typing 20% (Figure 4A). 
		NOTE: The stirring rate can be selected from 0% to 100%.</li>
		<li>Click on the door lock sector and set it to activate at temperatures exceeding 80 &#176;C (Figure 4B). 
		NOTE: This setting ensures automatic closure of the chamber door when the internal temperature surpasses 80 &#176;C.</li>
		<li>Click on the table icon on the top bar (Figure 5A) and set the temperature gradient (T1) to an extraction duration of 10 min, microwave power to 1800 W, and temperature to 120 &#176;C.</li>
		<li>Activate the stirrer by clicking on the stirrer button until the green light appears.</li>
		<li>Set the blower fan speed to level 3 (maximum) (Figure 5B).</li>
		<li>To hold the extraction time, select the desired extraction temperature (T2) by setting the extraction duration to 15 min, the microwave power to 1800 W, and the temperature to 120 &#176;C.</li>
		<li>Set the stirrer and fan speed as stated in section 2.2.6 and 2.2.7 (Figure 5A,B). 
		NOTE: The maximum temperature and microwave power are 260 &#176;C and 1800 W.</li>
		<li>Set the cooling time by clicking on the cooling button at the lower left corner of the screen and selecting the duration of 10 min (Figure 6).</li>
		<li>Save the method by clicking on the save icon at the top right corner of the screen (Figure 7A).</li>
		<li>Ensure that after saving the method conditions, the extraction conditions graph will be displayed on the screen with the play button in the lower right corner (Figure 7B).</li>
		<li>Start the extraction process by choosing the number of vessels used (Figure 7C). 
		NOTE: Up to 15 vessels can be used in one extraction, and if the desired number of vessels is utilized, ensure the balanced placement of vessels in the chamber.</li>
		<li>Following extraction, centrifuge the extracts at 4 &#176;C, 9072 x g, for 15 min, using a refrigerated centrifuge machine.</li>
		<li>Collect the supernatant with a 10 mL glass pipette (Figure 8) and store it at -20 &#176;C in the freezer for further study. 
		NOTE: Depending on the particle size and density of the plant residue, the extracts will require longer centrifugation times (20-30 min).</li>
	</ol>
	</li>
</ol>
3. Determination of phenolic compounds
<ol>
	<li>Determination of total phenolic content
	<ol>
		<li>Determine the total phenolic content of the CS extracts by referencing the protocol with some modifications17.</li>
		<li>Prepare a 10-fold dilution of samples by diluting with distilled water.</li>
		<li>Mix 10 &#181;L of the diluted sample with 20 &#181;L of undiluted Folin-Ciocalteu&#39;s reagent and allow them to react for 3 min.</li>
		<li>Next, add 100 &#181;L of 7.5% Na2CO3 solution to the mixture in each well of a 96-well plate.</li>
		<li>Prepare different concentrations for the gallic acid standard concentration range (please see Table 1 and Table 2) by diluting with distilled water.</li>
		<li>Mix them with 20 &#181;L of Folin-Ciocalteu&#39;s reagent and allow them to react for 3 min.</li>
		<li>Next, add 100 &#181;L of 7.5% Na2CO3 solution to the mixture in each well of a 96-well plate.</li>
		<li>Incubate the reaction for 30 min in the dark at room temperature.</li>
		<li>Measure the absorbance of the reaction solution at 765 nm using a microplate reader (Figure 9A).</li>
		<li>Plot the standard calibration curve using the concentrations of the standard and absorbance at 765 nm (Figure 10A).</li>
		<li>Express the results as mg of gallic acid equivalent (GAE) per g of the sample, and calculate using the following equation18: 
		NOTE: mg of gallic acid equivalent (GAE) per g of sample = [((A765 - c) / m)) in &#181;g gallic acid equivalent &#215; Total volume in reaction well (mL) x Dilution &#215; weight of dry sample (1 g) x Resultant volume of extract (mL)] / [(Volume of sample added into each well (mL) x Actual weight of dry sample (g) &#215; conversion factor from &#181;g to mg (1000)] 
		&#8203;where, c = y-intercept, m = slope</li>
	</ol>
	</li>
	<li>Determination of the total flavonoid content
	<ol>
		<li>Determine the total flavonoid content of the CS extract according to the protocol with some modifications17.</li>
		<li>Prepare a 5-fold dilution of samples by diluting with distilled water.</li>
		<li>Add 50 &#181;L of the diluted sample to 15 &#181;L of 5% NaNO2 and incubate in the dark for 5 min.</li>
		<li>Mix 15 &#181;L of 10% AlCl3 solution with the reaction and keep it at room temperature for 6 min.</li>
		<li>Then, add 100 &#181;L of 1 M NaOH solution to the reaction and incubate for a further 10 min.</li>
		<li>Measure the absorbance of the mixture at 510 nm (Figure 9B).</li>
		<li>Prepare different concentrations of quercetin standard range (please see Table 3 and 4) by adding them to 15 &#181;L of 5% NaNO2 and incubating in the dark for 5 min.</li>
		<li>Mix 15 &#181;L of 10% AlCl3 solution with the reaction and keep at room temperature for 6 min.</li>
		<li>Add 100 &#181;L of 1 M NaOH solution to the reaction and incubate further for 10 min.</li>
		<li>Determine the absorbance of the standard at 510 nm (Figure 9B).</li>
		<li>Plot the standard calibration curve using concentrations of the standard and absorbance at 510 nm (Figure 10B).</li>
		<li>Express the results as mg of quercetin equivalent (QE) per g of the sample, calculated by equation19 as follows: 
		NOTE: mg of quercetin equivalent (QE) per g of sample = [((A510 - c) / m)) in &#181;g quercetin equivalent &#215; Total volume in reaction well (mL) x Dilution &#215; weight of dry sample (1 g) x Resultant volume of extract (mL)] / [(Volume of sample added into each well (mL) x Actual weight of dry sample (g) &#215; conversion factor from &#181;g to mg (1000)] 
		where, c = y-intercept, m = slope</li>
	</ol>
	</li>
</ol>
4. Determination of the antioxidant activities
<ol>
	<li>1,1-Diphenyl-2-Picryl-Hydrazil (DPPH) radical scavenging assay
	<ol>
		<li>Determine the DPPH radical scavenging activity of CS extract according to the protocol with some modifications17.</li>
		<li>Prepare a 10-fold dilution of the samples by diluting them with distilled water.</li>
		<li>Mix 20 &#181;L of the diluted samples with 135 &#181;L of 0.1 mM DPPH solution.</li>
		<li>Prepare different concentrations of the Trolox standard concentration range (please see Table 5 and 6) by mixing with 135 &#181;L of 0.1 mM DPPH solution.</li>
		<li>Incubate the mixture in the dark at room temperature for 30 min.</li>
		<li>Measure the absorbance of the resultant at 517 nm (Figure 9C).</li>
		<li>Plot the standard calibration curve using concentrations of the standard and&#160;% inhibition (Figure 10C).</li>
		<li>Calculate the&#160;% inhibition of the DPPH assay as follows: 
		%&#160;Inhibition = [(absorbance of control &#8722; absorbance of sample)/ absorbance of control] &#215; 100</li>
		<li>Express the results as mg of the Trolox equivalent antioxidant capacity per g of the sample, calculated by the following equation20: 
		NOTE: mg of Trolox equivalent (TE) per g of sample = [((% inhibition-c) / m) in &#181;g Trolox equivalent &#215; Total volume in reaction well (mL) x Dilution &#215; weight of dry sample (1 g) x Resultant volume of extract (mL)] / [(Volume of sample added into each well (mL) x Actual weight of dry sample (g) &#215; conversion factor from &#181;g to mg (1000)] 
		&#8203;where, c = y-intercept, m = slope</li>
	</ol>
	</li>
	<li>2,2&#8242;-Azino-Bis-3-Ethylbenzthiazoline-6-Sulphonic acid (ABTS) radical scavenging assay
	<ol>
		<li>Determine the ABTS radical scavenging activity of CS extract using the protocol from the reference with some modifications17.</li>
		<li>Prepare the ABTS&#183;+ stock solution by mixing 7 mM ABTS and 2.45 mM potassium persulfate (1:2) and incubate in the dark at room temperature for 16 h.</li>
		<li>Prepare the working solution by mixing 5 mL of ABTS&#183;+ stock solution with 100 mL of deionized water.</li>
		<li>Mix 160 &#181;L of ABTS&#183;+ working solution with 10 &#181;L of the 10-fold diluted sample or Trolox standard at different concentrations (see Table 7&#160;and Table 8).</li>
		<li>Incubate the reaction in the dark at room temperature for 30 min.</li>
		<li>Determine the absorbance of the mixture at 734 nm (Figure 9D).</li>
		<li>Plot the standard calibration curve using concentrations of the standard and % inhibition (Figure 10D).</li>
		<li>Calculate % inhibition of the ABTS assay using the following formula: 
		%&#160;Inhibition = [(absorbance of control - absorbance of sample) / absorbance of control] &#215; 100.
		<ol>
			<li>Express the results as mg of the Trolox equivalent antioxidant capacity per g of the sample, calculated using the following equation21: 
			NOTE: mg of trolox equivalent (TE) per g of sample = [((%&#160;inhibition - c) / m)) in &#181;g Trolox equivalent &#215; Total volume in reaction well (mL) x Dilution &#215; weight of dry sample (1 g) x Resultant volume of extract (mL)] / [(Volume of sample added into each well (mL) x Actual weight of dry sample (g) &#215; conversion factor from &#181;g to mg (1000)] 
			&#8203;where, c = y-intercept, m = slope</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Ferric Reducing Antioxidant Power Assay (FRAP)
	<ol>
		<li>Determine the ferric-reducing antioxidant activity of CS extract according to the protocol with some modifications17.</li>
		<li>Prepare the FRAP reagent using a 30 mM acetate buffer at pH 3.6, which is a mixture of 10 mM TPTZ solution in 40 mM HCl and 20 mM FeCl3.6H2O solution at a ratio of 10:1:1.</li>
		<li>Place the FRAP reagent into an amber bottle until required. 
		NOTE: Ensure the FRAP reagent is brown. If contaminated by metal ions or other reactive compounds, the reagent will turn purple and must be discarded. Use only freshly prepared reagents.</li>
		<li>Prepare a 5-fold dilution of the samples by diluting them with distilled water.</li>
		<li>Add 10 &#181;L of the diluted sample or 20 &#181;L of FeSO4.7H2O at different standard concentrations (Table 9 and Table 10) to 180 &#181;L of the FRAP solution.</li>
		<li>Incubate the reaction at room temperature for 4 min.</li>
		<li>Evaluate the absorbance of the mixture at 593 nm (Figure 9E).</li>
		<li>Plot the standard calibration curve using concentrations of the standard and absorbance at 593 nm (Figure 10E).</li>
		<li>Express the results as mg of FeSO4 per g of the sample, calculated using the following equation21: 
		NOTE: mg FeSO4 equivalent (Fe (II) E) per g sample = [((A593 - c) / m)) in &#181;g FeSO4 equivalent &#215; Total volume in reaction well (mL) x Dilution &#215; weight of dry sample (1 g) x Resultant volume of extract (mL)] / [(Volume of sample added into each well (mL) x Actual weight of dry sample (g) &#215; conversion factor from &#181;g to mg (1000)] 
		&#8203;where, c = y-intercept, m = slope</li>
	</ol>
	</li>
	<li>Perform all assays (TPC, TFC, DPPH, ABTS, and FRAP) of each sample in triplicate. In this study, water was used as the blank for most assays, except DPPH, where ethanol served as the blank to address background absorbance.</li>
</ol>
5. Statistical analysis
<ol>
	<li>Use SPSS software to carry out a statistical analysis of the experimental data.</li>
	<li>Conduct the normality test using the Shapiro-Wilk test.</li>
	<li>Compare the bioactive substances and antioxidant activities of polyols-based MAE CS extract and conventional solvents-based MAE CS extracts using one-way ANOVA with Duncan&#39;s multiple range tests.</li>
	<li>Express all data as mean &#177; SD (n = 3) and define the significance level at p &#60; 0.05.</li>
</ol>

Polyol-Based Microwave-Assisted Extraction for Phenolic and Antioxidant Yields

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<li>Yu, M., Gouvinhas, I., Rocha, J., Barros, A. I. R. N. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Phytochemical+and+antioxidant+analysis+of+medicinal+and+food+plants+towards+bioactive+food+and+pharmaceutical+resources%5BTitle%5D)+AND+%22Sci+Rep%22%5BJournal%5D)">Phytochemical and antioxidant analysis of medicinal and food plants towards bioactive food and pharmaceutical resources.</a> Sci Rep. 11 (1), 10041(2021).</li>
<li>Lefebvre, T., Destandau, E., Lesellier, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Selective+extraction+of+bioactive+compounds+from+plants+using+recent+extraction+techniques%3A+A+review%5BTitle%5D)+AND+%22J+Chromatogr+A%22%5BJournal%5D)">Selective extraction of bioactive compounds from plants using recent extraction techniques: A review.</a> J Chromatogr A. 1635, 461770(2021).</li>
<li>Nandasiri, R., Eskin, N. M., Thiyam-Höllander, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Antioxidative+polyphenols+of+canola+meal+extracted+by+high+pressure%3A+Impact+of+temperature+and+solvents%5BTitle%5D)+AND+%22J+Food+Sci%22%5BJournal%5D)">Antioxidative polyphenols of canola meal extracted by high pressure: Impact of temperature and solvents.</a> J Food Sci. 84 (11), 3117-3128 (2019).</li>
<li>Jha, A. K., Sit, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Extraction+of+bioactive+compounds+from+plant+materials+using+combination+of+various+novel+methods%3A+A+review%5BTitle%5D)+AND+%22Trends+Food+Sci+Technol%22%5BJournal%5D)">Extraction of bioactive compounds from plant materials using combination of various novel methods: A review.</a> Trends Food Sci Technol. 119, 579-591 (2022).</li>
<li>Czarnecki, M. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Solvent+effect+on+the+competition+between+weak+and+strong+interactions+in+phenol+solutions+studied+by+near-infrared+spectroscopy+and+DFT+calculations%5BTitle%5D)+AND+%22Phys+Chem+Chem+Phys%22%5BJournal%5D)">Solvent effect on the competition between weak and strong interactions in phenol solutions studied by near-infrared spectroscopy and DFT calculations.</a> Phys Chem Chem Phys. 23 (35), 19188-19194 (2021).</li>
<li>Lu, W., Mackie, C. J., Xu, B., Head-Gordon, M., Ahmed, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+computational+and+experimental+view+of+hydrogen+bonding+in+glycerol+water+clusters%5BTitle%5D)+AND+%22J+Phys+Chem+A%22%5BJournal%5D)">A computational and experimental view of hydrogen bonding in glycerol water clusters.</a> J Phys Chem A. 126 (10), 1701-1710 (2022).</li>
<li>Fan, C., Liu, Y., Sebbah, T., Cao, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+theoretical+study+on+terpene-based+natural+deep+eutectic+solvent%3A+Relationship+between+viscosity+and+hydrogen-bonding+interactions%5BTitle%5D)+AND+%22Glob+Chall%22%5BJournal%5D)">A theoretical study on terpene-based natural deep eutectic solvent: Relationship between viscosity and hydrogen-bonding interactions.</a> Glob Chall. 5 (3), 2000103(2021).</li>
<li>Liese, S., Schlaich, A., Netz, R. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Dielectric+constant+of+aqueous+solutions+of+proteins+and+organic+polymers+from+molecular+dynamics+simulations%5BTitle%5D)+AND+%22J+Chem+Phys%22%5BJournal%5D)">Dielectric constant of aqueous solutions of proteins and organic polymers from molecular dynamics simulations.</a> J Chem Phys. 156 (22), 224903(2022).</li>
<li>Noreland, E., Gestblom, B., Sjöblom, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Dielectric+relaxation+studies+of+1-hexanol+and+1%2C+2-hexanediol+in+heptane%5BTitle%5D)+AND+%22J+Solution+Chem%22%5BJournal%5D)">Dielectric relaxation studies of 1-hexanol and 1, 2-hexanediol in heptane.</a> J Solution Chem. 18, 303-312 (1989).</li>
<li>Wohlfarth, C. Permittivity (dielectric constant) of liquids. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aWohlfarth%2C+C+%22CRC+Handbook+of+Chemistry+and+Physics%22">CRC Handbook of Chemistry and Physics</a>. 6, http://webdelprofesor.ula.ve/ciencias/isolda/libros/handbook.pdf (1994).</li>
<li>Dean, J. R. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aDean%2C+JR+%22Extraction+techniques+for+environmental+analysis%22">Extraction techniques for environmental analysis</a>. , John Wiley & Sons. (2022).</li>
<li>Nour, A. H., Oluwaseun, A. R., Nour, A. H., Omer, M. S., Ahmed, N. Microwave-assisted extraction of bioactive compounds. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aNour%2C+AH+%22Microwave+heating.+Electromagnetic+fields+causing+thermal+and+non-thermal+effects%22">Microwave heating. Electromagnetic fields causing thermal and non-thermal effects</a>. , https://www.intechopen.com/books/10089 1-31 (2021).</li>
<li>David, F., Ochiai, N., Sandra, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Stir+bar+sorptive+extraction%3A+A+versatile%2C+sensitive+and+robust+technique+for+targeted+and+untargeted+analyses%5BTitle%5D)+AND+%22Evolution+of+solid-phase+microextraction+technology%22%5BJournal%5D)">Stir bar sorptive extraction: A versatile, sensitive and robust technique for targeted and untargeted analyses.</a> Evolution of solid-phase microextraction technology. , https://www.intechopen.com/books/10089. (2023).</li>
<li>López-Fernández, O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Determination+of+polyphenols+using+liquid+chromatography-tandem+mass+spectrometry+technique+%28LC-MS%2FMS%29%3A+A+review%5BTitle%5D)+AND+%22Antioxidants%22%5BJournal%5D)">Determination of polyphenols using liquid chromatography-tandem mass spectrometry technique (LC-MS/MS): A review.</a> Antioxidants. 9 (6), 479(2020).</li>
</ol>

The authors have nothing to disclose.

Various factors play a crucial role in the successful implementation of MAE, such as the phytochemical content of plant components, extraction duration, temperature, microwave power, solid-liquid ratio, and solvent concentration13. Plants typically exhibit varying profiles of phytochemicals; hence, the selection of natural plants rich in antioxidants and phenolic compounds is essential23. Furthermore, distinct bioactive constituents display a variety of polarities depending on the solvent used. Likewise, solvents exhibit differing polarities. Given that the polarity of solvents plays a crucial role in determining the efficacy of bioactive compound extraction from raw materials, it is imperative that the solvent&#39;s polarity aligns with that of the targeted bioactive molecules24.
In this study, various polyols were utilized to extract polyphenols and antioxidants from coffee silverskin using MAE. Polyphenols are predominantly polar, and solvents with high polarity typically enhance the yield of phenolic compounds25. Compared to samples with water and ethanol, those employing different polyols demonstrated higher efficiency in all measured responses, including TPC, TFC, and antioxidant assays such as DPPH, ABTS, and FRAP. Previous studies support the findings that aqueous-polyol mixtures can enhance the extraction yield of bioactive compounds in comparison to aqueous-ethanol mixtures9,10,11. When comparing different polyols, samples with a specific aqueous-polyol system failed to yield the highest value. However, it is interesting to note that samples with aqueous 1,2-hexanediol yielded the highest values in TPC and TFC assays. Meanwhile, those with aqueous-hexylene glycol yielded the highest values in DPPH and FRAP assays, and aqueous-pentylene glycol extract exhibited the highest values in the ABTS assay. The variation in values obtained from different assays such as TPC, TFC, DPPH, ABTS, and FRAP within aqueous-polyols systems can be attributed to several factors, including the distinctive characteristics of the polyols employed. Polyols exhibit differences in viscosity, polarity, and boiling points, directly influencing their efficacy in extracting bioactive compounds from plant materials26. One potential explanation may be attributed to the principle of &#34;Like dissolves like&#34;, wherein the particular solvent system is the most appropriate for facilitating the mass transfer of particular bioactive compounds27. This underscores the importance of selecting a solvent with a polarity that matches that of the targeted bioactive compound.
Another possible reason might be the fact that the difference in the number of hydroxyl groups (-OH) present in the solvent significantly impacts the yield of phenolic compounds28. Among these polyols, only glycerin contains three -OH groups, while the remaining polyols in this study contain two -OH groups. Solvents with a higher number of -OH groups tend to exhibit higher viscosity compared to those with fewer29. Elevated viscosity can impede the efficient transfer of active compounds during the extraction process, thereby reducing the overall yield. Furthermore, the dielectric constant of a solvent, closely linked to its polarity, plays a crucial role in determining its ability to dissolve polar or nonpolar solutes. Solvents with higher dielectric constants are more adept at dissolving polar solutes, and those with lower better suited to nonpolar solutes30. Among polyols, glycerin exhibits a comparatively high dielectric constant of 41.14, while hexylene glycol, pentylene glycol, and 1,2-hexanediol demonstrate lower dielectric constants of 25.86, 17.31, and 15.45, respectively31,32. The findings of this study suggest that the bioactive compounds within the sample may encompass low-polar constituents.
Extraction efficiency can be enhanced by optimizing the selection and composition of solvents, and further experimentation may be required to identify the most suitable solvent system. Although the investigation exhibits potential, it is limited by its sole focus on microwave-assisted extraction with polyols and its restricted evaluation of other variables, including extraction duration, temperature, solvent concentration, solid-liquid ratio, and extraction power. In addition, a mechanistic study is necessary to understand how polyols function due to their varied dielectric constants, directly impacting their solubility of polar or nonpolar solutes. Differences in dielectric constants among polyols highlight the importance of investigating their specific mechanisms in extracting bioactive compounds. Such research would offer valuable insights into solvent-solute interactions, aiding the optimization and selection of solvent systems for efficient extraction processes.
Regarding the MAE process, there are some limitations. While MAE can provide rapid heating, precise temperature control may be challenging, potentially leading to overheating and the degradation of thermally sensitive compounds33. However, the microwave power setting for gradient and extraction temperature can be set to the same microwave power to avoid temperature frustration during extraction. Additionally, MAE has limitations over heat-sensitive plant components. However, advanced Ethos X MAE technology can minimize the risk of deterioration by supporting effective heating at a shorter duration using dielectric heating34. Each vessel of the MAE chamber has its own limited maximum solid and liquid capacity. The solid-liquid ratio over this limitation can also greatly influence the concentration of the extracts11. The dissolution between solvent and solute can be promoted using stirrer bars, potentially leading to effective extraction and higher yield35. Furthermore, the extracted phenolic and flavonoid compounds within the extracts can be verified through additional analysis, such as Liquid Chromatography Triple Quadrupole Mass Spectrometry (LC-QQQ) and Liquid Chromatography Quadrupole Time-of-Flight Mass Spectrometry (LC-QTOF), to establish the presence of specific bioactive compounds and their respective quantities36.
The extraction of polyphenols and antioxidants from coffee silverskin using aqueous polyols through MAE demonstrated higher efficiency in comparison to water and aqueous-ethanol extracts. Based on the outcomes derived from the polyols-based MAE of CS extracts, it has been observed that the employment of the aqueous-hexylene glycol, aqueous-1,2-hexanediol, and aqueous-pentylene glycol systems resulted in significantly higher extraction yields of bioactive compounds and antioxidant capacities. Moreover, these findings underscore the potential of utilizing such extracted compounds for subsequent investigative analyses. The utilization of polyols as green solvents for bioactive compound extraction from plant materials through MAE promises environmental benefits and enhanced bioactive compound yield, offering a sustainable approach with the potential for use in cosmetic applications.

Nowadays, there is a global trend towards environmental awareness in the beauty industry, leading manufacturers to focus on green technology for extracting plant components using sustainable alternatives1. Typically, traditional solvents such as ethanol, methanol, and hexane are used to extract plant phenolic components and natural antioxidants2. Nevertheless, the presence of solvent residues within plant extracts poses a potential risk to human health, inducing skin and eye irritation3, particularly concerning their intended application in cosmetics. Consequently, it is challenging to eliminate such solvent residues from the extracts, a process that demands considerable investment in time, energy, and human resources4. Recently, superheated water, ionic liquids, deep eutectic solvents, and bio-derived solvents have emerged as promising approaches for green solvent extraction5. However, their use is still limited by product separation in aqueous-based processes. To address these challenges, the development of ready-to-use extracts emerges as a viable solution6.
Polyols are often used in cosmetic formulations as humectants because of their good polarity and ability to retain moisture from the environment7. In addition, polyols such as glycerin, propylene glycol, butylene glycol, methylpropanediol, isopentyldiol, pentylene glycol, 1,2-hexanediol, and hexylene glycol can be utilized for plant extractions. They are considered non-toxic, biodegradable, environmentally friendly, non-reactive, and safe solvents for use in plant extraction8. Additionally, polyols can withstand the heat generated during microwave-assisted extraction (MAE) due to their elevated boiling points and polarity9. These polyols are generally recognized as safe (GRAS) chemicals by the United States Food and Drug Administration (FDA). Unlike conventional solvents such as ethanol or methanol, which may require rigorous removal from the extract due to their potentially harmful effects, polyols offer the advantage of minimizing the energy, time, and costs associated with solvent removal processes10. This not only streamlines the extraction process but also enhances the overall efficiency and sustainability of the extraction method. Previous investigations have employed polyols such as propylene glycol and butylene glycol as solvents in the extraction of bioactive compounds from Camellia sinensis flowers10 and coffee pulp11, revealing significant potential for their role as sustainable alternative solvents in the plant extraction process. Thus, the continued development and optimization of a polyols-water solvent system holds the potential for significant advancements in green chemistry and sustainable industrial practices.
Generally, bioactive compounds found in plants are synthesized as secondary metabolites. These compounds can be categorized into three primary groups: terpenes and terpenoids, alkaloids, and phenolic compounds12. Various extraction methods are utilized under different conditions to isolate specific bioactive compounds from plants. Bioactive compounds from plant materials can be extracted using either conventional or non-conventional techniques. Traditional methods include maceration, reflux extraction, and hydro-distillation, while non-conventional methods consist of ultrasound-assisted extraction, enzyme-assisted extraction, microwave-assisted extraction (MAE), pulsed electric field-assisted extraction, supercritical fluid extraction, and pressurized liquid extraction13. These non-conventional methods are designed to enhance safety by utilizing safer solvents and auxiliaries, improving energy efficiency, preventing degradation of the bioactive components, and reducing environmental pollution14.
Furthermore, MAE is among the sophisticated green technologies for extracting bioactive compounds from plants. Conventional extraction procedures require significant amounts of time, energy, and high temperatures, which over time might degrade heat-sensitive bioactive compounds13. In contrast to conventional thermal extractions, MAE facilitates the extraction of bioactive compounds by generating localized heating within the sample, disrupting cell structures, and enhancing mass transfer, thereby increasing the efficiency of compound extraction. Heat is transferred from inside the plant cells by microwaves, which operate on the water molecules within the plant components13. Moreover, MAE has advanced to improve the extraction and separation of active compounds, increasing product yield, enhancing extraction efficiency, requiring fewer chemicals, and saving time and energy while preventing the destruction of bioactive compounds15.
This research focuses on the extraction of plant phenolic compounds and natural antioxidants through microwave-assisted extraction (MAE) using different types of polyols as solvents. The total phenolic content (TPC), total flavonoid content (TFC), and antioxidant activities (DPPH, ABTS, and FRAP) of polyol-based MAE extracts are determined. Additionally, polyol-based MAE is compared with MAE using conventional solvents such as water and ethanol. This research is expected to contribute to the development of environmentally sustainable extraction technology for natural components, promoting sustainability by reducing reliance on hazardous chemicals, shortening processing times, and minimizing energy consumption in raw material production for potential applications within the cosmetics industry.

<table><tbody><tr><td>1,2-Hexanediol</td><td>Chanjao Longevity Co., Ltd.</td><td></td><td></td></tr><tr><td>2,2 -Azino-bis 3 ethylbenzothiazoline-6-sulfonic acid diammonium salt (ABTS)</td><td>Sigma</td><td>A1888</td><td></td></tr><tr><td>2,2-Diphenyl-1-picrylhydrazyl (DPPH)</td><td>Sigma</td><td>D9132</td><td></td></tr><tr><td>2,4,6-Tri(2-pyridyl)-s-triazine (TPTZ)</td><td>Sigma</td><td>93285</td><td></td></tr><tr><td>2-Digital balance</td><td>Ohaus</td><td></td><td>Pioneer</td></tr><tr><td>4-Digital balance</td><td>Denver</td><td>SI-234</td><td></td></tr><tr><td>6-hydroxy-2,5,7,8 tetramethylchroman -2-carboxylic acid (Trolox)</td><td>Sigma</td><td>238813</td><td></td></tr><tr><td>96-well plate</td><td>SPL Life Science</td><td></td><td></td></tr><tr><td>Absolute ethanol</td><td>RCI Labscan</td><td>64175</td><td></td></tr><tr><td>Acetic acid</td><td>RCI Labscan</td><td>64197</td><td></td></tr><tr><td>Aluminum chloride</td><td>Loba Chemie</td><td>898</td><td></td></tr><tr><td>Automatic pipette</td><td>Labnet</td><td></td><td>Biopett</td></tr><tr><td>Butylene glycol</td><td>Chanjao Longevity Co., Ltd.</td><td></td><td></td></tr><tr><td>Ethos X advanced microwave extraction</td><td>Milestone Srl, Sorisole, Italy</td><td></td><td></td></tr><tr><td>Ferrous sulfate</td><td>Ajex Finechem</td><td>3850</td><td></td></tr><tr><td>Folin-Ciocalteu&#039;s reagent</td><td>Loba Chemie</td><td>3870</td><td></td></tr><tr><td>Freezer SF</td><td>Sanyo</td><td>C697(GYN)</td><td></td></tr><tr><td>Gallic acid</td><td>Sigma</td><td>398225</td><td></td></tr><tr><td>Grinder</td><td>Ou Hardware Products Co.,Ltd</td><td></td><td></td></tr><tr><td>Hexylene glycol</td><td>Chanjao Longevity Co., Ltd.</td><td></td><td></td></tr><tr><td>Hydrochloric acid (37%)</td><td>RCI Labscan</td><td>AR1107</td><td></td></tr><tr><td>Iron (III) chloride</td><td>Loba Chemie</td><td>3820</td><td></td></tr><tr><td>Isopentyldiol</td><td>Chanjao Longevity Co., Ltd.</td><td></td><td></td></tr><tr><td>Methanol</td><td>RCI Labscan</td><td>67561</td><td></td></tr><tr><td>Methylpropanediol&amp;nbsp;</td><td>Chanjao Longevity Co., Ltd.</td><td></td><td></td></tr><tr><td>Pentylene glycol</td><td>Chanjao Longevity Co., Ltd.</td><td></td><td></td></tr><tr><td>Potassium persulfate</td><td>Loba Chemie</td><td>5420</td><td></td></tr><tr><td>Propylene glycol</td><td>Chanjao Longevity Co., Ltd.</td><td></td><td></td></tr><tr><td>Quercetin</td><td>Sigma</td><td>Q4951</td><td></td></tr><tr><td>Refrigerated centrifuge</td><td>Hettich</td><td></td><td></td></tr><tr><td>Sodium acetate</td><td>Loba Chemie</td><td>5758</td><td></td></tr><tr><td>Sodium carbonate</td><td>Loba Chemie</td><td>5810</td><td></td></tr><tr><td>Sodium hydroxide</td><td>RCI Labscan</td><td>AR1325</td><td></td></tr><tr><td>Sodium nitrite</td><td>Loba Chemie</td><td>5954</td><td></td></tr><tr><td>SPECTROstar Nano microplate reader</td><td>BMG- LABTECH</td><td></td><td></td></tr><tr><td>SPSS software</td><td>IBM SPSS Statistics 20</td><td></td><td></td></tr><tr><td>Tray dryer</td><td>France Etuves</td><td>XUE343</td><td></td></tr></tbody></table>

microwave-assisted extraction of phenolic compounds and antioxidants for cosmetic applications using polyol-based technology

The utilization of polyols as green solvents for extracting bioactive compounds from plant materials has gained attention due to their safety and inert behavior with plant bioactive chemicals. This study explores the sustainable extraction of phenolic compounds and natural antioxidants from coffee silverskin using the microwave-assisted extraction (MAE) method with polyol-based solvents: glycerin, propylene glycol (PG), butylene glycol (BG), methylpropanediol (MPD), isopentyldiol (IPD), pentylene glycol, 1,2-hexanediol, and hexylene glycol (HG). A comparative analysis was conducted on conventional and non-conventional solvent extractions, focusing on their impact on the bioactive compounds of MAE, encompassing parameters such as total phenolic content (TPC), total flavonoid content (TFC), and antioxidant activities like the 1,1-diphenyl-2-picrylhydrazyl radical scavenging assay (DPPH), the 2,2&#8242;-azino-bis(-3-ethylbenzothiazoline-6-sulphonic acid) radical scavenging assay (ABTS), and the ferric reducing antioxidant power assay (FRAP). The highest values were observed for TPC with aqueous-1,2-hexanediol extraction (52.0 &#177; 3.0 mg GAE/g sample), TFC with aqueous-1,2-hexanediol extraction (20.0 &#177; 1.7 mg QE/g sample), DPPH with aqueous-HG&#160;extraction (13.6 &#177; 0.3 mg TE/g sample), ABTS with aqueous-pentylene glycol extraction (8.2 &#177; 0.1 mg TE/g sample), and FRAP with aqueous-HG extraction (21.1 &#177; 1.3 mg Fe (II) E/g sample). This research aims to advance eco-friendly extraction technology through natural plant components, promoting sustainability by minimizing hazardous chemical use while reducing time and energy consumption, with potential applications in cosmetics.

Effect of polyols solvents and conventional solvents on total phenolic content, total flavonoid content, DPPH, FRAP, and ABTS antioxidant assays 
Solvent polarity should be compatible with that of targeted active molecules to improve the extraction efficiency of bioactive substances from plants22. Experiments were conducted using various solvents (water, ethanol, glycerin, propylene glycol, butylene glycol, methylpropanediol, isopentyldiol, pentylene glycol, 1,2-hexanediol, and hexylene glycol) to assess their impact on the bioactive compounds and antioxidant activities of MAE coffee silverskin extract.
Effect of polyols solvents and conventional solvents on total phenolic content 
The total phenolic content of each extraction with different solvents was analyzed. The highest phenolic content was yielded in samples with aqueous-1,2-hexanediol (52.0 &#177; 3.0 mg GAE/g sample), while the lowest TPC was revealed in samples with water extraction (31.4 &#177; 4.3 mg GAE/g sample), and these values were significantly different from those of all other conditions. The samples with aqueous-pentylene glycol yielded the second-highest TPC value, followed by samples with aqueous-butylene glycol, methylpropanediol, and other solvent systems (Figure 11A). When comparing samples with conventional solvents (water and aqueous-ethanol system) and samples with polyols-based solvents, significant differences in TPC values can be observed (p &#60; 0.05).
Effect of polyols solvents and conventional solvents on total flavonoid content 
The total flavonoid content of each extraction with different solvents was analyzed. The highest flavonoid content was yielded in samples with aqueous-1,2-hexanediol (20.0 &#177; 1.7 mg QE/g sample), demonstrating a significant difference from that of all other extracts. The samples with aqueous-isopentydiol revealed the lowest TFC value (8.8 &#177; 0.7 mg QE/g sample), which was not significantly different from&#160;aqueous-methyl propanediol, and aqueous-ethanol extracts. Moreover, the second highest TFC value was found in the sample with aqueous-pentylene glycol, followed by aqueous-hexylene glycol, aqueous-propylene glycol, aqueous-butylene glycol, and aqueous-glycerin (Figure 11B).
Effect of polyols solvents and conventional solvents on antioxidant assays 
The antioxidant activities of the extracts with polyols and conventional solvents were evaluated using DPPH, ABTS, and FRAP assays. The highest value for the DPPH assay was measured in samples with aqueous-hexylene glycol (13.6 &#177; 0.3 mg TE/g sample) and the lowest in samples with aqueous-ethanol (4.5 &#177; 0.2 mg GAE/g sample), and these values were significantly different from other extracts (p &#60; 0.05). The second highest DPPH values were observed in samples with aqueous-1,2-hexanediol, followed by aqueous-pentylene glycol, aqueous-methyl propanediol, and other solvent systems (Figure 11C).
The highest ABTS value was measured in samples with aqueous-pentylene glycol (8.2 &#177; 0.1 mg TE/g sample) and the lowest in samples with water (5.6 &#177; 0.04 mg GAE/g sample), and these values were significantly different from other extracts (p &#60; 0.05). The second highest ABTS values were detected in aqueous-butylene glycol and aqueous-1,2-hexanediol, followed by samples with aqueous-glycerin, aqueous-methyl propanediol, and other solvent systems (Figure 11D).
The highest FRAP values were observed in samples with aqueous-hexylene glycol (21.1 &#177; 1.3 mg Fe (II) E/g sample and the lowest in water extraction (11.5 &#177; 0.2 Fe (II) E/g sample), with these values being significantly different (p &#60; 0.05) for the remaining solvents. Moreover, the second highest FRAP values were found in samples with aqueous-pentylene glycol, followed by aqueous-butylene glycol, aqueous-glycerin, and other solvent systems (Figure 11E).
When comparing the antioxidant activities of samples with conventional solvents (water and aqueous ethanol), those containing polyols exhibited significantly higher antioxidant activities in all antioxidant assays (DPPH, ABTS, and FRAP) (p &#60; 0.05).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/67033/67033fig01.jpg" /> 
Figure 1: Reaction in experimental containers and the MAE chamber. (A)&#160;Sample and solvent are added to the white inter-layer vessel of a Teflon container before extraction. (B)&#160;Each container is placed inside the microwave chamber before starting the extraction. <a href="https://www.jove.com/files/ftp_upload/67033/67033fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/67033/67033fig02.jpg" /> 
Figure 2: Special tools for closing the reaction vessels. After adding the sample and solvent to the Teflon container, the lids are applied to the top of the container, placed in the vessel holder, and fastened tightly using the tools. <a href="https://www.jove.com/files/ftp_upload/67033/67033fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/67033/67033fig03.jpg" /> 
Figure 3: Extraction method. (A)&#160;Extraction method, created by entering the method section. (B) The SK eT accessory is applied for the MAE process. <a href="https://www.jove.com/files/ftp_upload/67033/67033fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/67033/67033fig04.jpg" /> 
Figure 4: Stirring rate and door lock function setting. (A)&#160;The magnetic stirrer bars inside each vessel can be activated by choosing the stirring rate. (B)&#160;The door lock function limits the temperature, allowing the chamber to be opened after extraction. <a href="https://www.jove.com/files/ftp_upload/67033/67033fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/67033/67033fig05.jpg" /> 
Figure 5: Setting the extraction conditions. (A) Entering the table icon and setting the extraction conditions such as time, temperature, and microwave power. (B) Opening the stirrer button and choosing the blower speed. <a href="https://www.jove.com/files/ftp_upload/67033/67033fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/67033/67033fig06.jpg" /> 
Figure 6: Setting the cooling time. Applying the cooling time to reduce the inside temperature in the MAE chamber. <a href="https://www.jove.com/files/ftp_upload/67033/67033fig06large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/67033/67033fig07.jpg" /> 
Figure 7: Starting the extraction process. (A) Saving the method created for extraction. (B) Clicking the play icon to start the extraction process. (C) Choosing the number of vessels to start the extraction. <a href="https://www.jove.com/files/ftp_upload/67033/67033fig07large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 8" class="xfigimg" src="/files/ftp_upload/67033/67033fig08.jpg" /> 
Figure 8: Picture of the final extract after extraction using MAE. Obtaining the supernatant after centrifuging. <a href="https://www.jove.com/files/ftp_upload/67033/67033fig08large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 9" class="xfigimg" src="/files/ftp_upload/67033/67033fig09.jpg" /> 
Figure 9: The 96 well-plates for determining the TPC, TFC, DPPH scavenging activity, ABTS scavenging activity and FRAP assay of the extracts. (A) Determining the TPC for the gallic acid standard plate from a concentration of 2.5-75 &#181;g/mL and sample extracts. (B) Determining the TFC for the quercetin standard plate from concentrations of 2.5-50&#160;&#181;g/mL and TFC assay to measure sample extracts. (C) Determining the DPPH scavenging activity&#160;for the Trolox standard plate from concentrations of 0.25-12.5 &#181;g/mL and the DPPH scavenging activity&#160;detection plate of sample extracts. (D) Determining the ABTS scavenging activity&#160;for the Trolox standard plate from concentrations of 0.25-5 &#181;g/mL and the ABTS scavenging activity&#160;detection plate of sample extracts. (E) Determining the FRAP assay&#160;for the FeSO4 standard plate from concentrations of 0.25-10 &#181;g/mL and the FRAP assay&#160;detection plate of sample extracts. <a href="https://www.jove.com/files/ftp_upload/67033/67033fig09large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 10" class="xfigimg" src="/files/ftp_upload/67033/67033fig10.jpg" /> 
Figure 10: The standard calibration curves for the TPC, TFC, DPPH scavenging activity, ABTS scavenging activity, and FRAP assay. (A) The standard curve for determining TPC, plotted by concentrations of the gallic acid standard and absorbance at A765. (B) The standard curve for determining TFC, plotted by concentrations of the quercetin standard and absorbance at A510. (C) The standard curve for determining DPPH scavenging activity, plotted by concentrations of the Trolox standard and % inhibition. (D) The standard curve for determining ABTS scavenging activity, plotted by concentrations of the Trolox standard and % inhibition. (E) The standard curve for measuring the FRAP assay, plotted by concentrations of the ferrous sulfate standard and absorbance at A593. <a href="https://www.jove.com/files/ftp_upload/67033/67033fig10large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 11" class="xfigimg" src="/files/ftp_upload/67033/67033fig11.jpg" /> 
Figure 11: The effect of solvent types on the TPC, TFC, DPPH scavenging activity, ABTS scavenging activity, and FRAP assay in the MAE&#160;of coffee silverskin. (A) The effect of solvent types on total phenolic content. (B) The effect of solvent types on total flavonoid content. (C) The effect of solvent types on DPPH scavenging activity. (D) The effect of solvent types on ABTS scavenging activity. (E) The effect of solvent types on the FRAP assay. Values are indicated as Mean &#177; SD (n = 3). Values with different superscript letters express a statistically significant difference (p &#60; 0.05). <a href="https://www.jove.com/files/ftp_upload/67033/67033fig11large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Table 1: Gallic acid standard curve preparation. Preparing the standard concentration range of 2.5-75 &#181;g/mL in the 96-well plate. B = blank, 1-7 = number of wells on the 96-well plate. <a href="https://www.jove.com/files/ftp_upload/67033/Table 1 Gallic acid standard curve preparation.xlsx" target="_blank">Please click here to download this Table.</a>
Table 2: Final concentration calculation of gallic acid standards. Preparing the standard concentration range of 2.5-75&#160;&#181;g/mL. Final concentrations (&#181;g/mL) of gallic acid are calculated accordingly. Final concentration (&#181;g/mL) = (Initial concentration (mg/ mL) &#215; Initial volume (&#181;L) / final volume (&#181;L)) &#215; (1000 &#181;g/1 mg). <a href="https://www.jove.com/files/ftp_upload/67033/Table 2 Final concentration gallic acid.xlsx" target="_blank">Please click here to download this Table.</a>
Table 3: Quercetin standard curve preparation. Preparing the standard concentration range of 2.5-50 &#181;g/mL in the 96-well plate. B = blank, 1-7 = number of wells on the 96-well plate. <a href="https://www.jove.com/files/ftp_upload/67033/Table 3 Quercetin standard curve preparation.xlsx" target="_blank">Please click here to download this Table.</a>
Table 4: Final concentration calculation table for quercetin standards. Preparing the standard concentration range of 2.5-50&#160;&#181;g/mL. Final concentrations (&#181;g/mL) of quercetin are calculated accordingly. Final concentration (&#181;g/mL) = (Initial concentration (mg/mL) &#215; Initial volume (&#181;L) / final volume (&#181;L)) &#215; (1000 &#181;g/1 mg). <a href="https://www.jove.com/files/ftp_upload/67033/Table 4 Final concentration quercetin.xlsx" target="_blank">Please click here to download this Table.</a>
Table 5: Trolox standard curve preparation in the concentration range of 0.25-12.5 &#181;g/mL. Preparing the standard concentration range of 0.25-12.5 &#181;g/mL in the 96-well plate. B = blank, C = control, 1-7 = number of wells on the 96-well plate. <a href="https://www.jove.com/files/ftp_upload/67033/Table 5 Trolox standard curve preparation.xlsx" target="_blank">Please click here to download this Table.</a>
Table 6: Final concentration calculation of the Trolox standards for DPPH assay. Preparing the standard concentration range of 0.25-12.5 &#181;g/mL including the final concentrations (&#181;g/mL) of Trolox. Final concentration (&#181;g/mL) = (Initial concentration (mg/mL) &#215; Initial volume (&#181;L) / final volume (&#181;L)) &#215; (1000 &#181;g/1 mg). <a href="https://www.jove.com/files/ftp_upload/67033/Table 6 Final concentration for trolox DPPH.xlsx" target="_blank">Please click here to download this Table.</a>
Table 7: Trolox standard curve preparation in the concentration range of 0.25-5 &#181;g/mL. Preparing the stock standard concentration range of 0.25-5 &#181;g/mL in the 96-well plate. B = blank, C = control, 1-7 = number of wells on the 96-well plate. <a href="https://www.jove.com/files/ftp_upload/67033/Table 7 Trolox standard curve preparation.xlsx" target="_blank">Please click here to download this Table.</a>
Table 8: Final concentration calculation of the Trolox standards for ABTS assay. Preparing the standard concentration range of 0.25-5 &#181;g/mL, including the final concentrations (&#181;g/mL) of Trolox. Final concentration (&#181;g/mL) = (Initial concentration (mg/mL) &#215; Initial volume (&#181;L) / final volume (&#181;L)) &#215; (1000 &#181;g/1 mg). <a href="https://www.jove.com/files/ftp_upload/67033/Table 8 Final concentration for trolox ABTS.xlsx" target="_blank">Please click here to download this Table.</a>
Table 9: Final concentration calculation table for the FeSO4 standards. Preparing the preparation of standard concentration range of 2.5-100 &#181;g/mL, including the final concentrations (&#181;g/mL) of FeSO4. Final concentration (&#181;g/mL) = (Initial concentration (mg/mL) &#215; Initial volume (&#181;L) / final volume (&#181;L)) &#215; (1000 &#181;g/1 mg). <a href="https://www.jove.com/files/ftp_upload/67033/Table 9 Final concentration for FeSO4.xlsx" target="_blank">Please click here to download this Table.</a>
Table 10: FeSO4 standard curve preparation. Preparing the standard concentration range of 0.25-10 &#181;g/mL in the 96-well plate. B = blank. <a href="https://www.jove.com/files/ftp_upload/67033/Table 10 FeSO4 standard curve preparation.xlsx" target="_blank">Please click here to download this Table.</a>

Author Spotlight: Eco-Friendly Extraction of Bioactive Compounds Using Polyol-Based Microwave-Assisted Techniques

This protocol details the utilization of a polyol-based microwave-assisted extraction method for extracting phenolic compounds and natural antioxidants, representing a practical and environmentally sustainable approach to the development of ready-to-use extracts.

Microwave-Assisted Extraction of Phenolic Compounds and Antioxidants for Cosmetic Applications Using Polyol-Based Technology

The utilization of polyols as green solvents for extracting bioactive compounds from plant materials has gained attention due to their safety and inert ...

microwave-assisted-extraction-phenolic-compounds-antioxidants-for

Universidad Científica del Sur

Research

JoVE Journal

Chemistry

1.5K Views.  Mae Fah Laung University. This protocol details the utilization of a polyol-based microwave-assisted extraction method for extracting phenolic compounds and natural antioxidants, representing a practical and environmentally sustainable approach to the development of ready-to-use extracts.

Video: Author Spotlight: Eco-Friendly Extraction of Bioactive Compounds Using Polyol-Based Microwave-Assisted Techniques

Microwave-assisted Intramolecular Dehydrogenative Diels-Alder Reactions for the Synthesis of Functionalized Naphthalenes/Solvatochromic Dyes

Functionalized naphthalenes have applications in a variety of research fields ranging from the synthesis of natural or biologically active molecules to the preparation of new organic dyes. Although numerous strategies have been reported to access naphthalene scaffolds, many procedures still present limitations in terms of incorporating functionality, which in turn narrows the range of available substrates. The development of versatile methods for direct access to substituted naphthalenes is therefore highly desirable.
The Diels-Alder (DA) cycloaddition reaction is a powerful and attractive method for the formation of saturated and unsaturated ring systems from readily available starting materials. A new microwave-assisted intramolecular dehydrogenative DA reaction of styrenyl derivatives described herein generates a variety of functionalized cyclopenta[b]naphthalenes that could not be prepared using existing synthetic methods. When compared to conventional heating, microwave irradiation accelerates reaction rates, enhances yields, and limits the formation of undesired byproducts.
The utility of this protocol is further demonstrated by the conversion of a DA cycloadduct into a novel solvatochromic fluorescent dye via a Buchwald-Hartwig palladium-catalyzed cross-coupling reaction. Fluorescence spectroscopy, as an informative and sensitive analytical technique, plays a key role in research fields including environmental science, medicine, pharmacology, and cellular biology. Access to a variety of new organic fluorophores provided by the microwave-assisted dehydrogenative DA reaction allows for further advancement in these fields.

Microwave-assisted intramolecular dehydrogenative Diels-Alder (DA) reactions provide concise access to functionalized cyclopenta[b]naphthalene building blocks. The utility of this methodology is demonstrated by one-step conversion of the dehydrogenative DA cycloadducts into novel solvatochromic fluorescent dyes via Buchwald-Hartwig palladium-catalyzed cross-coupling reactions.

Functionalized naphthalenes have applications in a variety of research fields ranging from the synthesis of natural or biologically active molecules to ...

Preparation of Keratin Hydrolysate from Chicken Feathers and Its Application in Cosmetics

Keratin hydrolysates (KHs) are established standard components in hair cosmetics. Understanding the moisturizing effects of KH is advantageous for skin-care cosmetics. The goals of the protocol are: (1) to process chicken feathers into KH by alkaline-enzymatic hydrolysis and purify it by dialysis, and (2) to test if adding KH into an ointment base (OB) increases hydration of the skin and improves skin barrier function by diminishing transepidermal water loss (TEWL). During alkaline-enzymatic hydrolysis feathers are first incubated at a higher temperature in an alkaline environment and then, under mild conditions, hydrolyzed with proteolytic enzyme. The solution of KH is dialyzed, vacuum dried, and milled to a fine powder. Cosmetic formulations comprising from oil in water emulsion (O/W) containing 2, 4, and 6 weight% of KH (based on the weight of the OB) are prepared. Testing the moisturizing properties of KH is carried out on 10 men and 10 women at time intervals of 1, 2, 3, 4, 24, and 48 h. Tested formulations are spread at degreased volar forearm sites. The skin hydration of stratum corneum (SC) is assessed by measuring capacitance of the skin, which is one of the most world-wide used and simple methods. TEWL is based on measuring the quantity of water transported per a defined area and period of time from the skin. Both methods are fully non-invasive. KH makes for an excellent occlusive; depending on the addition of KH into OB, it brings about a 30% reduction in TEWL after application. KH also functions as a humectant, as it binds water from the lower layers of the epidermis to the SC; at the optimum KH addition in the OB, up to 19% rise in hydration in men and 22% rise in women occurs.

The goal of the protocol is to prepare keratin hydrolysate from chicken feathers by alkaline-enzymatic hydrolysis and to test whether adding keratin hydrolysate into a cosmetics ointment base improves skin barrier function (heightening hydration and diminishing transepidermal water loss). Tests are conducted on men and woman volunteers.

Keratin hydrolysates (KHs) are established standard components in hair cosmetics. Understanding the moisturizing effects of KH is advantageous for ...

Biochemistry

Purification and Visualization of Lipopolysaccharide from Gram-negative Bacteria by Hot Aqueous-phenol Extraction

Lipopolysaccharide (LPS) is a major component of Gram-negative bacterial outer membranes. It is a tripartite molecule consisting of lipid A, which is embedded in the outer membrane, a core oligosaccharide and repeating O-antigen units that extend outward from the surface of the cell1, 2. LPS is an immunodominant molecule that is important for the virulence and pathogenesis of many bacterial species, including Pseudomonas aeruginosa, Salmonella species, and Escherichia coli3-5, and differences in LPS O-antigen composition form the basis for serotyping of strains. LPS is involved in attachment to host cells at the initiation of infection and provides protection from complement-mediated killing; strains that lack LPS can be attenuated for virulence6-8. For these reasons, it is important to visualize LPS, particularly from clinical isolates. Visualizing LPS banding patterns and recognition by specific antibodies can be useful tools to identify strain lineages and to characterize various mutants. 
In this report, we describe a hot aqueous-phenol method for the isolation and purification of LPS from Gram-negative bacterial cells. This protocol allows for the extraction of LPS away from nucleic acids and proteins that can interfere with visualization of LPS that occurs with shorter, less intensive extraction methods9. LPS prepared this way can be separated by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) and directly stained using carbohydrate/glycoprotein stains or standard silver staining methods. Many anti-sera to LPS contain antibodies that cross-react with outer membrane proteins or other antigenic targets that can hinder reactivity observed following Western immunoblot of SDS-PAGE-separated crude cell lysates. Protease treatment of crude cell lysates alone is not always an effective way of removing this background using this or other visualization methods. Further, extensive protease treatment in an attempt to remove this background can lead to poor quality LPS that is not well resolved by any of the aforementioned methods. For these reasons, we believe that the following protocol, adapted from Westpahl and Jann10, is ideal for LPS extraction.

We describe a modified hot aqueous-phenol extraction method for purifying lipopolysaccharide (LPS) from Gram-negative bacteria. Once extracted, the LPS can be subsequently analyzed by SDS-PAGE and visualized by direct staining or Western immunoblot.

Lipopolysaccharide (LPS)  is a major component of Gram-negative bacterial outer membranes. It is a tripartite molecule consisting of  lipid A, which is ...

Immunology and Infection

Synthesis of Plant Phenol-derived Polymeric Dyes for Direct or Mordant-based Hair Dyeing

Effective hair dyeing through in situ incubation of keratin hair with the products of fungal laccase-catalyzed polymerization of plant phenols has been previously demonstrated. However, the dyeing process takes a long time to complete compared to commercial hair-dyeing products. To overcome this bottleneck, pre-synthesized polymeric products of the oxidative reaction of Trametes versicolor laccase on catechin and catechol, either with or without mordant agents (e.g., FeSO4), were here employed to achieve permanent keratin hair dyeing in various colors and shades. The laccase action in acidic sodium acetate buffer led to a deep black coloration after coupling reactions between the plant phenols. The colored dye products were then desalted and concentrated with ultrafiltration. The dyes, with or without mordant agents, caused a significant increase in &#916;E values (i.e., color difference value) in gray human hair within 2.5 hours. In addition, different keratin colors and shades were induced depending upon the mordanting and pH changes. The dyed hair also exhibited a strong resistance to detergent treatments, indicating that our methods can give rise to permanent hair dyeing.&#160;Overall, our work has provided novel insight into developing eco-friendly hair-dyeing methods as alternatives to commercial toxic diamine-based dyes.

Here, we present a protocol to use pre-synthesized polymeric products derived from fungal laccase-catalyzed polymerization of plant phenols, either with or without mordant agents (e.g., FeSO4), to induce detergent-resistant keratin hair dyeing within 2.5 hours.

Effective hair dyeing through in situ incubation of keratin hair with the products of fungal laccase-catalyzed polymerization of plant phenols has been ...

Extraction and Purification of Polyphenols from Freeze-dried Berry Powder for the Treatment of Vascular Smooth Muscle Cells In Vitro

Epidemiological studies indicate that increased flavonoid intake correlates with decreased mortality due to cardiovascular diseases (CVD) in the United States (US) and Europe. Berries are widely consumed in the US and have a high polyphenolic content. Polyphenols have been shown to interact with many molecular targets and to exert numerous positive biological functions, including antioxidant, anti-inflammatory, and cardioprotective effects. Polyphenols isolated from blackberry (BL), raspberry (RB), and black raspberry (BRB) reduce oxidative stress and cellular senescence in response to angiotensin II (Ang II). This work provides a detailed description of the protocol used to prepare the polyphenol extracts from freeze-dried berries. Polyphenol extractions from freeze-dried berry powder were performed using 80% aqueous ethanol and an ultrasonic-assisted extraction method. The crude extract was further purified and fractionated using chloroform and ethyl acetate, respectively. The effects of both crude and purified extracts were tested on Vascular Smooth Muscle Cells (VSMCs) in culture.

Extraction and Purification of Polyphenols from Freeze-dried Berry Powder for the Treatment of Vascular Smooth Muscle Cells In Vitro

This work details a step-by-step method to prepare polyphenol-rich extracts from freeze-dried berry powder. In addition, it provides a thorough description of how to use these polyphenol-rich extracts in cell culture in the presence of the peptide hormone angiotensin II (Ang II) using Vascular Smooth Muscle Cells (VSMCs).

Epidemiological studies indicate that increased flavonoid intake correlates with decreased mortality due to cardiovascular diseases (CVD) in the United ...

Employing Pressurized Hot Water Extraction (PHWE) to Explore Natural Products Chemistry in the Undergraduate Laboratory

A recently developed pressurized hot water extraction (PHWE) method which utilizes an unmodified household espresso machine to facilitate natural products research has also found applications as an effective teaching tool. Specifically, this technique has been used to introduce second- and third-year undergraduates to aspects of natural products chemistry in the laboratory. In this report, two experiments are presented: the PHWE of eugenol and acetyleugenol from cloves and the PHWE of seselin and (+)-epoxysuberosin from the endemic Australian plant species Correa reflexa. By employing PHWE in these experiments, the crude clove extract, enriched in eugenol and acetyleugenol, was obtained in 4-9% w/w from cloves by second-year undergraduates and seselin and (+)-epoxysuberosin were isolated in yields of up to 1.1% w/w and 0.9% w/w from C. reflexa by third-year students. The former exercise was developed as a replacement for the traditional steam distillation experiment providing an introduction to extraction and separation techniques, while the latter activity featured guided-inquiry teaching methods in an effort to simulate natural products bioprospecting. This primarily derives from the rapid nature of this PHWE technique relative to traditional extraction methods that are often incompatible with the time constraints associated with undergraduate laboratory experiments. This rapid and practical PHWE method can be used to efficiently isolate various classes of organic molecules from a range of plant species. The complementary nature of this technique relative to more traditional methods has also been demonstrated previously.

Employing Pressurized Hot Water Extraction PHWE to Explore Natural Products Chemistry in the Undergraduate Laboratory

Here, we employ a pressurized hot water extraction (PHWE) method, which utilizes an unmodified household espresso machine to introduce undergraduates to natural products chemistry in the laboratory. Two experiments are presented: PHWE of eugenol and acetyleugenol from cloves and PHWE of seselin and (+)-epoxysuberosin from the Australian plant Correa reflexa.

A recently developed pressurized hot water extraction (PHWE) method which utilizes an unmodified household espresso machine to facilitate natural products ...

Ultrafast Lignin Extraction from Unusual Mediterranean Lignocellulosic Residues

Pretreatment is still the most expensive step in lignocellulosic biorefinery processes. It must be made cost-effective by minimizing chemical requirements as well as power and heat consumption and by using environment-friendly solvents. Deep eutectic solvents (DESs) are key, green, and low-cost solvents in sustainable biorefineries. They are transparent mixtures characterized by low freezing points resulting from at least one hydrogen bond donor and one hydrogen bond acceptor. Although DESs are promising solvents, it is necessary to combine them with an economic heating technology, such as microwave irradiation, for competitive profitability. Microwave irradiation is a promising strategy to shorten the heating time and boost fractionation because it can rapidly attain the appropriate temperature. The aim of this study was to develop a one-step, rapid method for biomass fractionation and lignin extraction using a low-cost and biodegradable solvent. 
In this study, a microwave-assisted DES pretreatment was conducted for 60 s at 800 W, using three kinds of DESs. The DES mixtures were facilely prepared from choline chloride (ChCl) and three hydrogen-bond donors (HBDs): a monocarboxylic acid (lactic acid), a dicarboxylic acid (oxalic acid), and urea. This pretreatment was used for biomass fractionation and lignin recovery from marine residues (Posidonia leaves and aegagropile), agri-food byproducts (almond shells and olive pomace), forest residues (pinecones), and perennial lignocellulosic grasses (Stipa tenacissima). Further analyses were conducted to determine the yield, purity, and molecular weight distribution of the recovered lignin. In addition, the effect of DESs on the chemical functional groups in the extracted lignin was determined by Fourier-transform infrared (FTIR) spectroscopy. The results indicate that the ChCl-oxalic acid mixture affords the highest lignin purity and the lowest yield. The present study demonstrates that the DES-microwave process is an ultrafast, efficient, and cost-competitive technology for lignocellulosic biomass fractionation.

Deep eutectic solvent-based, microwave-assisted pretreatment is a green, fast, and efficient process for lignocellulosic fractionation and high-purity lignin recovery.

Pretreatment is still the most expensive step in lignocellulosic biorefinery processes. It must be made cost-effective by minimizing chemical requirements ...

Semi-Targeted Ultra-High-Performance Chromatography Coupled to Mass Spectrometry Analysis of Phenolic Metabolites in Plasma of Elderly Adults

A group of 23 elderly persons was given functional meals (a beverage and a muffin) specially formulated for the prevention of sarcopenia (age-related loss of muscle mass). Plasma samples were taken at the beginning of the intervention and after 30 days of consuming the functional meals. A semi-targeted ultra-high-performance chromatography coupled with tandem mass (UPLC-MS/MS) analysis was carried out to identify phenolic compounds and their metabolites. Plasma proteins were precipitated with ethanol and the samples were concentrated and resuspended in the mobile phase (1:1 acetonitrile: water) before injection into the UPLC-MS/MS instrument. Separation was carried out with a C18 reverse-phase column, and compounds were identified using their experimental mass, isotopic distribution, and fragment pattern. Compounds of interest were compared to those of data banks and the internal semi-targeted library. Preliminary results showed that the major metabolites identified after the intervention were phenylacetic acid, glycitin, 3-hydroxyphenylvaleric acid, and gomisin M2.

The goal of this protocol is to detect phenolic metabolites in plasma using a semi-targeted chromatography-mass spectrometry method.

A group of 23 elderly persons was given functional meals (a beverage and a muffin) specially formulated for the prevention of sarcopenia (age-related loss ...

A Generalized Method for Determining Free Soluble Phenolic Acid Composition and Antioxidant Capacity of Cereals and Legumes

Phenolic acids are a class of organic compounds that bear both a phenolic group, and a carboxylic group. They are found in grains and concentrate in the bran of cereals or seed coat of legumes. They possess antioxidant properties that have generated much research interest in recent years, about their potential antioxidant protective health functions. This work presents a generalized method for the extraction of free soluble phenolic acids from whole grains and analysis of their antioxidant capacity. Five whole grain samples comprising two cereals (wheat and yellow corn) and three legumes (cowpea bean, kidney bean, and soybean), were used. The grains were milled into flour and their free soluble phenolic acids extracted using aqueous methanol. The compounds were then identified using a high-pressure liquid chromatograph (HPLC). The Folin-Ciocalteu method was used to determine their total phenolic content while their antioxidant capacities were determined using the DPPH radical scavenging capacity, Trolox equivalent antioxidant capacity (TEAC) and oxygen radical absorbance capacity (ORAC) assays. The phenolic acids identified included vanillic, caffeic, p-coumaric and ferulic acids. Vanillic acid was identified only in cowpea while caffeic acid was identified only in kidney bean. p-Coumaric acid was identified in yellow corn, cowpea, and soybean, while ferulic acid was identified in all the samples. Ferulic acid was the predominant phenolic acid identified. The total concentration of phenolic acids in the samples decreased in the following order: soybean &gt; cowpea bean &gt; yellow corn = kidney bean &gt; wheat. The total antioxidant capacity (sum of values of DPPH, TEAC and ORAC assays) decreased as follows: soybean &gt; kidney bean &gt; yellow corn = cowpea bean &gt; wheat. This study concluded that HPLC analysis as well as DPPH, TEAC, and ORAC assays provide useful information about the phenolic acid composition and antioxidant properties of whole grains.

Phenolic acids are important phytochemicals that are present in whole grains. They possess bioactive properties such as antioxidant protective functions. This work aimed at reporting on a generalized method for the HPLC identification, total phenolic content estimation, and determination of the antioxidant capacity of phenolic acids in cereals and legumes.

Phenolic acids are a class of organic compounds that bear both a phenolic group, and a carboxylic group. They are found in grains and concentrate in the ...

Using Multiple Light Scattering to Examine the Stability of Phyllanthus emblica L. Extracts Obtained with Different Extraction Methods

The extraction intermediate of traditional Chinese medicine is the key intermediate in the preparation process, and its stability has an important impact on the effectiveness and quality of the final product. However, existing stability evaluation methods are often time-consuming and labor-intensive, requiring long-term observation and the operation of complex equipment (such as high-performance liquid chromatography), and it is difficult to obtain more physical information about the instability of the system. Therefore, there is an urgent need to establish a fast and accurate stability analysis technology for traditional Chinese medicine. Multiple light scattering is a cutting-edge analytical method that can accurately and rapidly evaluate the stability of traditional Chinese medicines in an environment-friendly manner without changing the nature or state of the sample or using organic reagents.
In this work, using the precise scanning data of multiple light scattering, the present protocol rapidly acquired the variation curves for layer thickness, particle migration speed, and average particle size over time. This enabled the precise identification of the mechanism and crucial characteristics causing the system&#39;s instability in its early stages. Of note, the research period for the extraction process can be considerably shortened by the detailed quantification of the system stability, which also allows for a quick, accurate, and in-depth analysis of the effects of various extraction processes on the stability of Phyllanthus emblica L.

Using Multiple Light Scattering to Examine the Stability of Phyllanthus emblica L. Extracts Obtained with Different Extraction Methods

Here, we introduce a stability evaluation method based on multiple light scattering technology to evaluate the stability of traditional Chinese medicine extracts.

The extraction intermediate of traditional Chinese medicine is the key intermediate in the preparation process, and its stability has an important impact ...

Microwave-Assisted Extraction of Phenolic Compounds and Antioxidants for Cosmetic Applications Using Polyol-Based Technology

Summary

Explore More Videos

Microwave-assisted Intramolecular Dehydrogenative Diels-Alder Reactions for the Synthesis of Functionalized Naphthalenes/Solvatochromic Dyes

Preparation of Keratin Hydrolysate from Chicken Feathers and Its Application in Cosmetics

Purification and Visualization of Lipopolysaccharide from Gram-negative Bacteria by Hot Aqueous-phenol Extraction

Synthesis of Plant Phenol-derived Polymeric Dyes for Direct or Mordant-based Hair Dyeing

Extraction and Purification of Polyphenols from Freeze-dried Berry Powder for the Treatment of Vascular Smooth Muscle Cells In Vitro

Employing Pressurized Hot Water Extraction (PHWE) to Explore Natural Products Chemistry in the Undergraduate Laboratory

Ultrafast Lignin Extraction from Unusual Mediterranean Lignocellulosic Residues

Semi-Targeted Ultra-High-Performance Chromatography Coupled to Mass Spectrometry Analysis of Phenolic Metabolites in Plasma of Elderly Adults

A Generalized Method for Determining Free Soluble Phenolic Acid Composition and Antioxidant Capacity of Cereals and Legumes

Using Multiple Light Scattering to Examine the Stability of Phyllanthus emblica L. Extracts Obtained with Different Extraction Methods