Name: Autor em destaque: extração ecológica de compostos bioativos usando técnicas assistidas por microondas à base de poliol
Uploaded: 2024-08-23T13:40:00
Description: The utilization of polyols as green solvents for extracting bioactive compounds from plant materials has gained attention due to their safety and inert ...

Este estudo foi financiado pela Universidade Mae Fah Luang. Os autores gostariam de agradecer ao Instituto de Chá e Café da Universidade Mae Fah Luang por facilitar a conexão entre os pesquisadores e os agricultores locais em relação à aquisição de amostras de casca de prata de café.

Os detalhes dos reagentes e dos equipamentos utilizados neste estudo estão listados na Tabela de Materiais.
1. Preparação experimental
<ol><li>Preparação de amostras de plantas<ol><li>Recolher o café fresco (Coffea arabica) e secá-lo a 60 °C num secador de tabuleiros durante 72 h11.</li>
		<li>Moa a casca de prata do café seco (CS) em um pó fino usando um moedor e armazene-o em temperatura ambiente para análise pos...

Extração assistida por micro-ondas à base de poliol para rendimentos fenólicos e antioxidantes

<ol>
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Vários fatores desempenham um papel crucial na implementação bem-sucedida do MAE, como o conteúdo fitoquímico dos componentes da planta, duração da extração, temperatura, potência de micro-ondas, relação sólido-líquido e concentração de solvente13. As plantas normalmente exibem perfis variados de fitoquímicos; Assim, a seleção de plantas naturais ricas em antioxidantes e compostos fenólicos é essencial23. Além disso, constituintes bioativos distintos e...

Atualmente, há uma tendência global de conscientização ambiental na indústria da beleza, levando os fabricantes a se concentrarem em tecnologia verde para extração de componentes vegetais usando alternativas sustentáveis1. Normalmente, solventes tradicionais, como etanol, metanol e hexano, são usados para extrair componentes fenólicos vegetais e antioxidantes naturais2. No entanto, a presença de resíduos de solventes nos extratos vegetais representa um risco potencial para a saúde humana, induzindo irritação cutânea e ocular3, particularmente no que diz respeito à sua aplicação em cosméticos. Consequentemente, é desafiador eliminar esses resíduos de solventes dos extratos, um processo que demanda considerável investimento em tempo, energia e recursos humanos4. Recentemente, água superaquecida, líquidos iônicos, solventes eutéticos profundos e solventes bioderivados surgiram como abordagens promissoras para a extração de solventes verdes5. No entanto, seu uso ainda é limitado pela separação do produto em processos aquosos. Para enfrentar esses desafios, o desenvolvimento de extratos prontos para uso surge como uma solução viável6.
Os polióis são frequentemente usados em formulações cosméticas como umectantes devido à sua boa polaridade e capacidade de reter a umidade do ambiente7. Além disso, polióis como glicerina, propilenoglicol, butilenoglicol, metilpropanodiol, isopentildiol, pentilenoglicol, 1,2-hexanodiol e hexilenoglicol podem ser utilizados para extrações de plantas. São considerados solventes não tóxicos, biodegradáveis, ecologicamente corretos, não reativos e seguros para uso na extração de plantas8. Além disso, os polióis podem suportar o calor gerado durante a extração assistida por micro-ondas (MAE) devido aos seus pontos de ebulição e polaridade elevados9. Esses polióis são geralmente reconhecidos como produtos químicos seguros (GRAS) pela Food and Drug Administration (FDA) dos Estados Unidos. Ao contrário dos solventes convencionais, como etanol ou metanol, que podem exigir uma remoção rigorosa do extrato devido aos seus efeitos potencialmente nocivos, os polióis oferecem a vantagem de minimizar a energia, o tempo e os custos associados aos processos de remoção de solventes10. Isso não apenas agiliza o processo de extração, mas também aumenta a eficiência geral e a sustentabilidade do método de extração. Investigações anteriores empregaram polióis como propilenoglicol e butilenoglicol como solventes na extração de compostos bioativos de flores de Camellia sinensis 10 e polpa de café11, revelando potencial significativo para seu papel como solventes alternativos sustentáveis no processo de extração de plantas. Assim, o desenvolvimento e a otimização contínuos de um sistema de solvente de polióis-água têm potencial para avanços significativos na química verde e em práticas industriais sustentáveis.
Geralmente, os compostos bioativos encontrados nas plantas são sintetizados como metabólitos secundários. Esses compostos podem ser categorizados em três grupos primários: terpenos e terpenóides, alcalóides e compostos fenólicos12. Vários métodos de extração são utilizados em diferentes condições para isolar compostos bioativos específicos das plantas. Compostos bioativos de materiais vegetais podem ser extraídos usando técnicas convencionais ou não convencionais. Os métodos tradicionais incluem maceração, extração de refluxo e hidrodestilação, enquanto os métodos não convencionais consistem em extração assistida por ultrassom, extração assistida por enzima, extração assistida por micro-ondas (MAE), extração assistida por campo elétrico pulsado, extração de fluido supercrítico e extração de líquido pressurizado13. Esses métodos não convencionais são projetados para aumentar a segurança, utilizando solventes e auxiliares mais seguros, melhorando a eficiência energética, evitando a degradação dos componentes bioativos e reduzindo a poluição ambiental14.
Além disso, o MAE está entre as sofisticadas tecnologias verdes para extração de compostos bioativos de plantas. Os procedimentos convencionais de extração requerem quantidades significativas de tempo, energia e altas temperaturas, que com o tempo podem degradar compostos bioativos sensíveis ao calor13. Em contraste com as extrações térmicas convencionais, o MAE facilita a extração de compostos bioativos, gerando aquecimento localizado dentro da amostra, interrompendo as estruturas celulares e aumentando a transferência de massa, aumentando assim a eficiência da extração do composto. O calor é transferido de dentro das células vegetais por microondas, que operam nas moléculas de água dentro dos componentes da planta13. Além disso, o MAE avançou para melhorar a extração e separação de compostos ativos, aumentando o rendimento do produto, aumentando a eficiência da extração, exigindo menos produtos químicos e economizando tempo e energia, evitando a destruição de compostos bioativos15.
Esta pesquisa se concentra na extração de compostos fenólicos vegetais e antioxidantes naturais por meio de extração assistida por micro-ondas (MAE) usando diferentes tipos de polióis como solventes. O conteúdo fenólico total (TPC), o conteúdo total de flavonóides (TFC) e as atividades antioxidantes (DPPH, ABTS e FRAP) dos extratos de MAE à base de poliol são determinados. Além disso, o MAE à base de poliol é comparado com o MAE usando solventes convencionais, como água e etanol. Espera-se que esta pesquisa contribua para o desenvolvimento de tecnologia de extração ambientalmente sustentável para componentes naturais, promovendo a sustentabilidade reduzindo a dependência de produtos químicos perigosos, encurtando os tempos de processamento e minimizando o consumo de energia na produção de matérias-primas para aplicações potenciais na indústria de cosméticos.

<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

A utilização de polióis como solventes verdes para extração de compostos bioativos de materiais vegetais tem ganhado atenção devido à sua segurança e comportamento inerte com produtos químicos bioativos vegetais. Este estudo explora a extração sustentável de compostos fenólicos e antioxidantes naturais da casca de café usando o método de extração assistida por micro-ondas (MAE) com solventes à base de poliol: glicerina, propilenoglicol (PG), butilenoglicol (BG), metilpropanodiol (MPD), isopentildiol (IPD), pentilenoglicol, 1,2-hexanodiol e hexilenoglicol (HG). Foi realizada uma análise comparativa de extrações com solventes convencionais e não convencionais, com foco em seu impacto nos compostos bioativos do MAE, englobando parâmetros como conteúdo fenólico total (TPC), teor total de flavonoides (TFC) e atividades antioxidantes como o ensaio de sequestro de radicais 1,1-difenil-2-picrilhidrazil (DPPH), o ensaio de sequestro de radicais 2,2′-azino-bis(-3-etilbenzotiazolina-6-ácido sulfônico) (ABTS) e o ensaio de poder antioxidante redutor férrico (FRAP). Os maiores valores foram observados para TPC com extração aquosa de 1,2-hexanodiol (amostra de 52,0 ± 3,0 mg GAE/g), TFC com extração aquosa de 1,2-hexanodiol (amostra de 20,0 ± 1,7 mg QE/g), DPPH com extração aquosa de HG (amostra de 13,6 ± 0,3 mg TE/g), ABTS com extração aquosa de pentilenoglicol (amostra de 8,2 ± 0,1 mg TE/g) e FRAP com extração aquosa de HG (amostra de 21,1 ± 1,3 mg Fe (II) E/g). Esta pesquisa visa avançar a tecnologia de extração ecologicamente correta por meio de componentes naturais de plantas, promovendo a sustentabilidade ao minimizar o uso de produtos químicos perigosos e reduzir o tempo e o consumo de energia, com aplicações potenciais em cosméticos.

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.

Autor em destaque: extração ecológica de compostos bioativos usando técnicas assistidas por microondas à base de poliol

Extração assistida por micro-ondas de compostos fenólicos e antioxidantes para aplicações cosméticas usando tecnologia baseada em poliol

This research talks about finding eco-friendly ways to extract plant bioactive compounds. We are testing how well microwave assisted extraction with the polyol-based solvents work for extracting plants bioactive substances, especially phenol compounds. Additionally, exploring this method could lead to more efficient and effective extraction technique for the cosmetic industries.Today's cosmetic research is also about using green technology and green chemistry. Nonconventional extraction techniques such as microwave-assist extraction, and extraction, have been interestingly employed to extract plants bioactive compounds. However, organic solvents are still commonly utilized for bioactive component extraction.Using polyol-based solvent for microwave assistant extraction turned out to be more effective than using traditional solvent like water and ethanol. The results show that the plant extract obtained with polyol have higher level of phenolic content for the flavanoid content and antioxidant activity. Compared to those extracted with conventional solvent.Our procedure stands out because it is beneficial, environmentally friendly and safe for human health. It is also more energy efficient and cost effective than traditional processes, making it an excellent alternative for sustainable and simple production. Our finding show that polyols combined with unconventional extraction methods can extract phenol compounds.The next challenge is whether this extraction method can be used to extract other plant, secondary metabolized, and if it can be commercialized to extract bioactive compounds for cosmetic purposes.

Efeito de solventes polióis e solventes convencionais sobre o conteúdo de fenólicos totais, teor de flavonóides totais, ensaios antioxidantes DPPH, FRAP e ABTS A polaridade do solvente deve ser compatível com a das moléculas ativas direcionadas para melhorar a eficiência de extração de substâncias bioativas das plantas22. Experimentos foram conduzidos usando vários solventes (água, etanol, glicerina, propilenoglicol, butilenoglicol, metilpropanodiol, isopentildiol, ...

Este protocolo detalha a utilização de um método de extração assistida por micro-ondas à base de poliol para extrair compostos fenólicos e antioxidantes naturais, representando uma abordagem prática e ambientalmente sustentável para o desenvolvimento de extratos prontos para uso.

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

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.

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.

Química

Extraction of Phenolic Compounds and Natural Antioxidants from Coffee Silverskin Using the Polyol-Based Microwave-Assisted Extraction Method

To begin, place the diluted 60%solvents for microwave assisted extraction on the working platform. Weigh 0.67 grams of the coffee silver skin and mix with 20 milliliters of extraction solvent at a one is to 30 ratio in a reaction container. Add a magnetic stir bar to the vessel to ensure uniform distribution of the heat and solvent within the sample.Close the vessel firmly with a special tool and place it into the microwave assisted extraction chamber. To set up the method, click on the toolbox icon on the top bar of the monitor and select the SK eT rotor in the accessory section. Then click on the stir and type 20%to set the stirring rate.Click on the door lock sector and set it to activate at temperatures exceeding 80 degrees Celsius. Then click on the table icon on the top bar and set the temperature gradient T1 to an extraction duration of 10 minutes. Microwave power to 1800 watts and temperature to 120 degrees Celsius.To activate the stir, click on the stir button and wait until the green light appears. Set the blower fan speed to level three. To hold the extraction time, select the desired extraction temperature T2 by setting the extraction duration to 15 minutes, the microwave power to 1800 watts and the temperature to 120 degrees Celsius.Now, click on the cooling button at the lower left corner of the screen and select the cooling time of 10 minutes. Click the Save icon at the top right corner of the screen. Start the extraction process by choosing the number of vessels used.Following extraction, centrifuge extracts at 9072g for 15 minutes at four degrees Celsius. Collect the supernatant with a 10 milliliter glass pipette and store it at minus 20 degrees Celsius. Dilute the coffee silver skin extracts tenfold with distilled water in a 96 well plate.Mix 10 microliters of the diluted sample with 20 microliters of undiluted folin-ciocalteu reagent, and allow them to react for three minutes. Next, add 100 microliters of 7.5%sodium carbonate solution to the mixture in each well of a 96 well plate. Prepare different concentrations for the gallic acid standard concentration range by diluting with distilled water.Mix them with 20 microliters of folin-ciocalteu reagent, and allow them to react for three minutes. Next, add 100 microliters of 7.5%sodium carbonate solution to the mixture in each well of a 96 well plate. Incubate the reaction for 30 minutes in the dark at room temperature.On a microplate reader, measure the absorbance of the reaction mixture at 765 nanometers. Plot the standard calibration curve using the concentrations of the standard and absorbance at 765 nanometers. Express the results as milligrams of gallic acid equivalent per gram of the sample.Aqueous 1, 2 hexanediol extraction yielded the highest total phenolic content and water extraction yielded the lowest. Aqueous 1, 2 hexanediol extraction yielded the highest total flavonoid content and aqueous isopentyldiol extraction yielded the lowest. Aqueous hexylene glycol extraction exhibited the highest DPPH assay value and aqueous ethanol extraction exhibited the lowest.Aqueous pentylene glycol extraction resulted in the highest ABTS assay value and water extraction resulted in the lowest. Aqueous hexylene glycol extraction showed the highest FRAP value and water extraction showed the lowest.