3.6. SFE–US with cosolvents
Table 3(C) shows that the addition of ethanol and water in SFE-US from the dried and milled material leads to higher extraction yields. This behavior is related to the polar nature of the chosen cosolvents, which allows extracting components that would notbe soluble in pure CO2, which is non polar. The molecules of the cosolvent compete with the active sites of the substrate to interact with the extractable compounds. Therefore, the presence of cosolvents helps breaking the interactions between substrate andsolute, which can be solubilized by the solvent mixture [7,38]. The increase of solubility caused by a cosolvent results from the formation of cosolvent–solute and solvent–cosolvent–solute groups[39]. A cosolvent with critical temperature lower than that of the supercritical fluid usually reduces the solubility of low volatility compounds, and the opposite occurs if the critical temperature of the cosolvent is higher [5]. Both cosolvents used in this work (ethanol and water) have critical temperatures higher than CO2(240.6◦C, 374.2◦C and 31.1◦C, respectively), so they enhance the solubility of many compounds present in the blackberry bagasse.An increase in SFE-US yield is observed when ethanol was usedas cosolvent, compared to water. Besides increasing yield, ethanolcan be removed easier than water, so its application is widely reported [40]. This behavior can be explained by the enhanced solubility of polar compounds in the mixture CO2+ ethanol. Moreover,the use of ethanol as cosolvent may have increased the numberof extracted compounds, thus reducing the selectivity of the process. Although water is more polar than ethanol, the SFE yields withwater as cosolvent were lower. Polarity is not the unique factoraffecting extraction yield. The type of interactions between solventand solute should also be comprehended.
As observed for SFE from blackberry bagasse, other works report remarkable enhancements in SFE yield by using ethanol as cosolvent at low concentrations. Luengthanaphol [41] compared yield and antioxidant activity of extracts (Tamarindus indica L.) from tamarind seeds obtained by SFE with pure CO2and with 10%ethanol as cosolvent, and verified that SFE of antioxidants is significantly improved with ethanol. Kitzberger et al. [42] observedan increase in the SFE yield from shiitake from 1.01% to 3.81% byusing 15% ethanol as cosolvent The contribution of ethanol and water as cosolvents in the recovery of phenolics, anthocyanins, and the antioxidant activity of the extracts can be observed in Fig. 3. In general, both cosolvents hadpositive influence on the extraction of the mentioned compounds,and the effect of water as cosolvent was clearly higher than ethanol.In SFE-US with cosolvents the highest anthocyanin content(17.54 ± 0.07 mg cyanidin 3-O-glucoside/g extract) was obtainedusing 5% water for the fresh sample. Tena et al. [43] and Murga et al.[44] reported that adding cosolvent to CO2 helps improving the extraction yield of some compounds, such as anthocyanins, phenolics, and antioxidant activity. It can be noted in Table 3(C) that the cosolvent ratio affects the anthocyanin concentration in the extracts, indicating that interactions between solute and substratemust have been broken and replaced by cosolvent molecules [7].Thus, the anthocyanin yield increased with the cosolvent concentration [45]. This is clear in the extractions from dried and crushed samples, where raising the water ratio from 5 to 10% strongly increased the anthocyanin concentration, due to the enhancement of the solute/cosolvent interactions that raise solubility [7]. The same effect is also noted in the extractions with ethanol, althoughthe anthocyanin recovery was lower than with water. Thus, wateras cosolvent not only increases SFE yield, but is also the most adequate solvent since is ecologically safe and cheap. Regarding phenolic compounds, the best results of SFE-US werefound using 10% water in the dried and crushed sample and 5%water in the fresh sample. As expected, water extracts phenolics efficiently, and the extracts with most phenolics also presented the highest antioxidant activities measured by DPPH and ABTS,evidencing some correlation between phenolics and antiradical capacity [46,47]. However, the relation shows that phenolics are not the unique responsible for the antioxidant activity of the extracts. Vegetable substrates contain several phenolic components with different antioxidant activities, and the synergism between antioxidants in a mixture makes their activity dependent of concentrations, structure and their chemical interactions [48].It can also be observed in Table 3(C) that the method ABTS ismore effective in the detection of antioxidant compounds than DPPH. The method DPPH is widely applied to determine the antioxidant activity in extracts and isolated compounds, such as phenolics, anthocyanins, flavonols and cumarins [49,50]. Every method provides precise and reproducible results, but the antioxidant activitiesmay differ significantly from one method to other. Thus, mostmethods provide partial results regarding antioxidant activity of complex extracts [51,52].The addition of water as cosolvent at 10% provides the highest antioxidant activities by both methods. For the extracts obtained by SFE-US with pure CO2 the antioxidant activities were quite lower,indicating again that this property is intimately related to phenolics, which are polar compounds that can hardly be extracted witha nonpolar solvent like CO2. Finally, evaluating the extracts obtained by SFE-US with cosolvent, one can conclude that to achieve the best yields it is preferable to use 10% ethanol, which provided yields eight times higher than with pure CO2. Since ethanol is a slightly polar solvent, its addition as cosolvent allowed the dissolution of polar substances that were not extracted with pure CO2. In terms of anthocyanins, phenolics, and antioxidant activity, it is better to use 10% water ascosolvent. The increase of such compounds may be due to the low solubility of water in CO2, which may lead to the coexistence of two phases. In this case, a liquid phase containing water as major component would help extracting preferentially phenolics and anthocyanins. Nevertheless, if SFE is performed on the freshsample, 5% water is more recommendable, since the water content of the sample seems to work as cosolvent and enhance the extraction of anthocyanins. Moreover, the recovery of anthocyaninscould have been enhanced due to pH reduction in the presence of CO2 and water, since anthocyanins are usually more stable in acidified media [53–55]. Summarizing, the differences between SFE-US with and without cosolvents can be attributed to the changes in cosolvent concentration, type of pretreatment, and extractionmethod [56].
3.6 การ SFE – สหรัฐฯ กับ cosolventsตารางแสดง 3(C) ที่แห่งเอทานอลและน้ำในสหรัฐอเมริกา SFE วัสดุแห้ง และสารนำไปสู่การแยกผลผลิตที่สูงขึ้น ปัญหานี้เกี่ยวข้องกับธรรมชาติของ cosolvents ท่าน ขั้วโลกซึ่งช่วยให้การดึงข้อมูลคอมโพเนนต์ที่จะ notbe ละลายใน CO2 บริสุทธิ์ ซึ่งไม่ใช่ ขั้วโลก โมเลกุลของ cosolvent ที่แข่งขันกับไซต์ใช้งานของพื้นผิวเพื่อโต้ตอบกับสาร extractable ดังนั้น สถานะของ cosolvents ช่วยทำลายการโต้ตอบระหว่างพื้นผิว andsolute ซึ่งสามารถ solubilized โดยผสมตัวทำละลาย [7,38] เพิ่มการละลายที่เกิดจากผลลัพธ์ cosolvent จากการก่อตัวของ cosolvent – ตัวถูกละลาย และตัวทำละลาย-cosolvent – ตัวกลุ่ม [39] Cosolvent ด้วยสำคัญอุณหภูมิที่ต่ำกว่าของเหลว supercritical มักจะลดความผันผวนต่ำสารละลาย และเกิดขึ้นตรงข้ามถ้าอุณหภูมิสำคัญของ cosolvent ที่ สูง [5] ทั้ง cosolvents ที่ใช้ในการทำงานนี้ (เอทานอลและน้ำ) ได้สูงกว่า CO2 อุณหภูมิสำคัญ (240.6◦C, 374.2◦C และ 31.1◦C ตามลำดับ), เพื่อ ให้พวกเขาเพิ่มการละลายของสารในชานอ้อย blackberry หลาย การเพิ่มผลผลิต SFE สหรัฐฯ จะสังเกตเมื่อเอทานอลถูก usedas cosolvent เปรียบเทียบกับน้ำ นอกจากการเพิ่มผลผลิต ethanolcan สามารถเอาออกได้ง่ายขึ้นกว่าน้ำ ดังนั้นโปรแกรมประยุกต์ของ รายงานอย่างกว้างขวาง [40] ลักษณะเช่นนี้สามารถอธิบายได้ โดยละลายเพิ่มสารโพลาร์ในผสม CO2 + เอทานอ นอกจากนี้ การใช้เอทานอลเป็น cosolvent อาจเพิ่มขึ้นสาร numberof สกัด ขจัดวิธีการ แม้ว่าน้ำจะขึ้นที่ขั้วโลกมากกว่าเอทานอล SFE ทำให้ withwater เป็น cosolvent ถูกล่าง ขั้วไม่พิมพ์แยกเฉพาะ factoraffecting ชนิดของการโต้ตอบระหว่างตัว solventand ควรยังสามารถ comprehendedAs observed for SFE from blackberry bagasse, other works report remarkable enhancements in SFE yield by using ethanol as cosolvent at low concentrations. Luengthanaphol [41] compared yield and antioxidant activity of extracts (Tamarindus indica L.) from tamarind seeds obtained by SFE with pure CO2and with 10%ethanol as cosolvent, and verified that SFE of antioxidants is significantly improved with ethanol. Kitzberger et al. [42] observedan increase in the SFE yield from shiitake from 1.01% to 3.81% byusing 15% ethanol as cosolvent The contribution of ethanol and water as cosolvents in the recovery of phenolics, anthocyanins, and the antioxidant activity of the extracts can be observed in Fig. 3. In general, both cosolvents hadpositive influence on the extraction of the mentioned compounds,and the effect of water as cosolvent was clearly higher than ethanol.In SFE-US with cosolvents the highest anthocyanin content(17.54 ± 0.07 mg cyanidin 3-O-glucoside/g extract) was obtainedusing 5% water for the fresh sample. Tena et al. [43] and Murga et al.[44] reported that adding cosolvent to CO2 helps improving the extraction yield of some compounds, such as anthocyanins, phenolics, and antioxidant activity. It can be noted in Table 3(C) that the cosolvent ratio affects the anthocyanin concentration in the extracts, indicating that interactions between solute and substratemust have been broken and replaced by cosolvent molecules [7].Thus, the anthocyanin yield increased with the cosolvent concentration [45]. This is clear in the extractions from dried and crushed samples, where raising the water ratio from 5 to 10% strongly increased the anthocyanin concentration, due to the enhancement of the solute/cosolvent interactions that raise solubility [7]. The same effect is also noted in the extractions with ethanol, althoughthe anthocyanin recovery was lower than with water. Thus, wateras cosolvent not only increases SFE yield, but is also the most adequate solvent since is ecologically safe and cheap. Regarding phenolic compounds, the best results of SFE-US werefound using 10% water in the dried and crushed sample and 5%water in the fresh sample. As expected, water extracts phenolics efficiently, and the extracts with most phenolics also presented the highest antioxidant activities measured by DPPH and ABTS,evidencing some correlation between phenolics and antiradical capacity [46,47]. However, the relation shows that phenolics are not the unique responsible for the antioxidant activity of the extracts. Vegetable substrates contain several phenolic components with different antioxidant activities, and the synergism between antioxidants in a mixture makes their activity dependent of concentrations, structure and their chemical interactions [48].It can also be observed in Table 3(C) that the method ABTS ismore effective in the detection of antioxidant compounds than DPPH. The method DPPH is widely applied to determine the antioxidant activity in extracts and isolated compounds, such as phenolics, anthocyanins, flavonols and cumarins [49,50]. Every method provides precise and reproducible results, but the antioxidant activitiesmay differ significantly from one method to other. Thus, mostmethods provide partial results regarding antioxidant activity of complex extracts [51,52].The addition of water as cosolvent at 10% provides the highest antioxidant activities by both methods. For the extracts obtained by SFE-US with pure CO2 the antioxidant activities were quite lower,indicating again that this property is intimately related to phenolics, which are polar compounds that can hardly be extracted witha nonpolar solvent like CO2. Finally, evaluating the extracts obtained by SFE-US with cosolvent, one can conclude that to achieve the best yields it is preferable to use 10% ethanol, which provided yields eight times higher than with pure CO2. Since ethanol is a slightly polar solvent, its addition as cosolvent allowed the dissolution of polar substances that were not extracted with pure CO2. In terms of anthocyanins, phenolics, and antioxidant activity, it is better to use 10% water ascosolvent. The increase of such compounds may be due to the low solubility of water in CO2, which may lead to the coexistence of two phases. In this case, a liquid phase containing water as major component would help extracting preferentially phenolics and anthocyanins. Nevertheless, if SFE is performed on the freshsample, 5% water is more recommendable, since the water content of the sample seems to work as cosolvent and enhance the extraction of anthocyanins. Moreover, the recovery of anthocyaninscould have been enhanced due to pH reduction in the presence of CO2 and water, since anthocyanins are usually more stable in acidified media [53–55]. Summarizing, the differences between SFE-US with and without cosolvents can be attributed to the changes in cosolvent concentration, type of pretreatment, and extractionmethod [56].
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