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

<ol>
	<li>Wawoczny, A., Gillner, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+most+potent+natural+pharmaceuticals,+cosmetics,+and+food+ingredients+isolated+from+plants+with+deep+eutectic+solvents.">The most potent natural pharmaceuticals, cosmetics, and food ingredients isolated from plants with deep eutectic solvents.</a> J Agric Food Chem. 71 (29), 10877-10900 (2023).</li><li>Syukur, M., Prahasiwi, M. S., Yuliani, S., Purwaningsih, Y., Indriyanti, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Profiling+of+active+compounds+of+extract+ethanol,+n-hexane,+ethyl+acetate+and+fraction+ethanol+of+star+anise+(Illicium+verum+hook.+F.)+and+determination+of+total+flavonoids,+total+phenolics+and+their+potential+as+antioxidants.">Profiling of active compounds of extract ethanol, n-hexane, ethyl acetate and fraction ethanol of star anise (Illicium verum hook. F.) and determination of total flavonoids, total phenolics and their potential as antioxidants.</a> Sci Technol Indones. 8 (2), 219-226 (2023).</li><li>Supjaroenporn, C., Khongcharoen, P., Myo, H., Khat-Udomkiri, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Studying+the+optimization,+characterization,+and+antioxidant+activities+of+phenolic+extracts+extracted+from+Rhus+chinensis+Mill.+Leaf+using+microwave-assisted+extraction+system+with+glycerol+as+a+green+solvent.">Studying the optimization, characterization, and antioxidant activities of phenolic extracts extracted from Rhus chinensis Mill. Leaf using microwave-assisted extraction system with glycerol as a green solvent.</a> Curr Bioact Compd. 20 (3), 68-82 (2024).</li><li>Gasser, M. S., Abdel Rahman, R. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sustainability+of+solvent+extraction+techniques+in+pollution+prevention+and+control.">Sustainability of solvent extraction techniques in pollution prevention and control.</a> Handbook of advanced approaches towards pollution prevention and control. , 33-66 (2021).</li><li>P&#322;otka-Wasylka, J., Rutkowska, M., Owczarek, K., Tobiszewski, M., Namie&#347;nik, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extraction+with+environmentally+friendly+solvents.">Extraction with environmentally friendly solvents.</a> TrAC, Trends Anal Chem. 91, 12-25 (2017).</li><li>Queffelec, J., Beraud, W., Torres, M. D., Dom&#237;nguez, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advances+in+obtaining+ready-to-use+extracts+with+natural+solvents.">Advances in obtaining ready-to-use extracts with natural solvents.</a> Sustain Chem Pharm. 38, 101478 (2024).</li><li>Can Karaca, A., Erdem, I. G., Ak, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+polyols+on+gelation+kinetics,+gel+hardness,+and+drying+properties+of+alginates+subjected+to+internal+gelation.">Effects of polyols on gelation kinetics, gel hardness, and drying properties of alginates subjected to internal gelation.</a> LWT. 92, 297-303 (2018).</li><li>Nastasi, J. R., Daygon, V. D., Kontogiorgos, V., Fitzgerald, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Qualitative+analysis+of+polyphenols+in+glycerol+plant+extracts+using+untargeted+metabolomics.">Qualitative analysis of polyphenols in glycerol plant extracts using untargeted metabolomics.</a> Metabolites. 13 (4), 566 (2023).</li><li>Khat-Udomkiri, N., Gatnawa, G., Boonlerd, N., Myo, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Valorization+of+Camellia+sinensis+flowers+in+cosmetic+and+pharmaceutical+applications:+Optimization+of+microwave-assisted+glycerin+extraction.">Valorization of Camellia sinensis flowers in cosmetic and pharmaceutical applications: Optimization of microwave-assisted glycerin extraction.</a> Waste Biomass Valori. 15, 323-335 (2023).</li><li>Myo, H., Yaowiwat, N., Pongkorpsakol, P., Aonbangkhen, C., Khat-Udomkiri, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Butylene+glycol+used+as+a+sustainable+solvent+for+extracting+bioactive+compounds+from+Camellia+sinensis+flowers+with+ultrasound-assisted+extraction.">Butylene glycol used as a sustainable solvent for extracting bioactive compounds from Camellia sinensis flowers with ultrasound-assisted extraction.</a> ACS omega. 8 (5), 4976-4987 (2023).</li><li>Myo, H., Khat-Udomkiri, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimization+of+ultrasound-assisted+extraction+of+bioactive+compounds+from+coffee+pulp+using+propylene+glycol+as+a+solvent+and+their+antioxidant+activities.">Optimization of ultrasound-assisted extraction of bioactive compounds from coffee pulp using propylene glycol as a solvent and their antioxidant activities.</a> Ultrason Sonochem. 89, 106127 (2022).</li><li>Twaij, B. M., Hasan, M. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bioactive+secondary+metabolites+from+plant+sources:+Types,+synthesis,+and+their+therapeutic+uses.">Bioactive secondary metabolites from plant sources: Types, synthesis, and their therapeutic uses.</a> Int J Plant Biol. 13 (1), 4-14 (2022).</li><li>Bitwell, C., Sen, I. S., Luke, C., Kakoma, M. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+review+of+modern+and+conventional+extraction+techniques+and+their+applications+for+extracting+phytochemicals+from+plants.">A review of modern and conventional extraction techniques and their applications for extracting phytochemicals from plants.</a> Sci Afr. 19, e01585 (2023).</li><li>Chakrabortty, S., Kumar, A., Patruni, K., Singh, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Recent advances in food biotechnology. , 353-370 (2022).</li><li>Fan, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanochemical+assisted+extraction+as+a+green+approach+in+preparation+of+bioactive+components+extraction+from+natural+products+-+A+review.">Mechanochemical assisted extraction as a green approach in preparation of bioactive components extraction from natural products - A review.</a> Trends Food Sci Technol. 129, 98-110 (2022).</li><li>Bessada, S. M., C Alves, R., Pp Oliveira, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coffee+silverskin:+A+review+on+potential+cosmetic+applications.">Coffee silverskin: A review on potential cosmetic applications.</a> Cosmetics. 5 (1), 5 (2018).</li><li>Myo, H., Nantarat, N., Khat-Udomkiri, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Changes+in+bioactive+compounds+of+coffee+pulp+through+fermentation-based+biotransformation+using+Lactobacillus+plantarum+TISTR+543+and+its+antioxidant+activities.">Changes in bioactive compounds of coffee pulp through fermentation-based biotransformation using Lactobacillus plantarum TISTR 543 and its antioxidant activities.</a> Fermentation. 7 (4), 292 (2021).</li><li>Molole, G. J., Gure, A., Abdissa, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+of+total+phenolic+content+and+antioxidant+activity+of+Commiphora+mollis+(oliv).+Engl.+Resin.">Determination of total phenolic content and antioxidant activity of Commiphora mollis (oliv). Engl. Resin.</a> BMC Chem. 16 (1), 48 (2022).</li><li>Barku, V., Opoku-Boahen, Y., Owusu-Ansah, E., Mensah, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antioxidant+activity+and+the+estimation+of+total+phenolic+and+flavonoid+contents+of+the+root+extract+of+Amaranthus+spinosus.">Antioxidant activity and the estimation of total phenolic and flavonoid contents of the root extract of Amaranthus spinosus.</a> Asian J Plant Sci Res. 3 (1), 69-74 (2013).</li><li>Samarasiri, M., Chandrasiri, T., Wijesinghe, D., Gunawardena, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antioxidant+capacity+and+total+phenolic+content+variations+against+Morinda+citrifolia+L.+fruit+juice+production+methods.">Antioxidant capacity and total phenolic content variations against Morinda citrifolia L. fruit juice production methods.</a> Int J Food Eng. 5, 293-299 (2019).</li><li>Rumpf, J., Burger, R., Schulze, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Statistical+evaluation+of+DPPH,+ABTS,+FRAP,+and+Folin-Ciocalteu+assays+to+assess+the+antioxidant+capacity+of+lignins.">Statistical evaluation of DPPH, ABTS, FRAP, and Folin-Ciocalteu assays to assess the antioxidant capacity of lignins.</a> Int J Biol Macromol. 233, 123470 (2023).</li><li>Lainez-Cer&#243;n, E., Ram&#237;rez-Corona, N., Jim&#233;nez-Mungu&#237;a, M. T., Palou, E., L&#243;pez-Malo, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extraction+of+bioactive+compounds+from+plants+by+means+of+new+environmentally+friendly+solvents.">Extraction of bioactive compounds from plants by means of new environmentally friendly solvents.</a> Research and technological advances in food science. , 301-332 (2022).</li><li>Yu, M., Gouvinhas, I., Rocha, J., Barros, A. I. R. N. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phytochemical+and+antioxidant+analysis+of+medicinal+and+food+plants+towards+bioactive+food+and+pharmaceutical+resources.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selective+extraction+of+bioactive+compounds+from+plants+using+recent+extraction+techniques:+A+review.">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-H&#246;llander, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antioxidative+polyphenols+of+canola+meal+extracted+by+high+pressure:+Impact+of+temperature+and+solvents.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extraction+of+bioactive+compounds+from+plant+materials+using+combination+of+various+novel+methods:+A+review.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Solvent+effect+on+the+competition+between+weak+and+strong+interactions+in+phenol+solutions+studied+by+near-infrared+spectroscopy+and+DFT+calculations.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+computational+and+experimental+view+of+hydrogen+bonding+in+glycerol+water+clusters.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+theoretical+study+on+terpene-based+natural+deep+eutectic+solvent:+Relationship+between+viscosity+and+hydrogen-bonding+interactions.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dielectric+constant+of+aqueous+solutions+of+proteins+and+organic+polymers+from+molecular+dynamics+simulations.">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., Sj&#246;blom, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dielectric+relaxation+studies+of+1-hexanol+and+1,+2-hexanediol+in+heptane.">Dielectric relaxation studies of 1-hexanol and 1, 2-hexanediol in heptane.</a> J Solution Chem. 18, 303-312 (1989).</li><li>Wohlfarth, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Permittivity+(dielectric+constant)+of+liquids.">Permittivity (dielectric constant) of liquids.</a> CRC Handbook of Chemistry and Physics. 6, (1994).</li><li>Dean, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Extraction techniques for environmental analysis. , (2022).</li><li>Nour, A. H., Oluwaseun, A. R., Nour, A. H., Omer, M. S., Ahmed, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microwave-assisted+extraction+of+bioactive+compounds.">Microwave-assisted extraction of bioactive compounds.</a> Microwave heating. Electromagnetic fields causing thermal and non-thermal effects. , 1-31 (2021).</li><li>David, F., Ochiai, N., Sandra, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stir+bar+sorptive+extraction:+A+versatile,+sensitive+and+robust+technique+for+targeted+and+untargeted+analyses.">Stir bar sorptive extraction: A versatile, sensitive and robust technique for targeted and untargeted analyses.</a> Evolution of solid-phase microextraction technology. , (2023).</li><li>L&#243;pez-Fern&#225;ndez, O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+of+polyphenols+using+liquid+chromatography-tandem+mass+spectrometry+technique+(LC-MS/MS):+A+review.">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...

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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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&#39;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&#160;</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, 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

Research

JoVE Journal

Chemistry

1.2K 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

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 ...