CET 97 DOI: 10.3303/CET2297093 Paper Received: 11 August 2022; Revised: 8 October 2022; Accepted: 22 November 2022 Please cite this article as: To D.V.T., Vu D., 2022, Codonopsis javanica Root Extraction with Enzyme Support, Chemical Engineering Transactions, 97, 553-558 DOI:10.3303/CET2297093 CHEMICAL ENGINEERING TRANSACTIONS VOL. 97, 2022 A publication of The Italian Association of Chemical Engineering Online at www.cetjournal.it Guest Editors: Jeng Shiun Lim, Nor Alafiza Yunus, Jiří Jaromír Klemeš Copyright © 2022, AIDIC Servizi S.r.l. ISBN 978-88-95608-96-9; ISSN 2283-9216 Codonopsis javanica Root Extraction with Enzyme Support Dien Vu Thanh Toa, Danh Vub* a Institute for Advanced Material Technology, Van Lang University, Ho Chi Minh city 700000, Vietnam b Institute of Applied Technology, Thu Dau Mot University, Binh Duong Province, Vietnam danhvc@tdmu.edu.vn Codonopsis javanica (CJ) contains various valuable bioactive compounds and is a cheaper medicinal herb than other ginsengs. In this study, Codonopsis javanica roots (CJR) were soaked in hot water in a thermostatic bath for extraction. Experiments were performed based on a change in the amount of enzyme α-amylase, extraction temperature and investigation time. Polyphenol, saponin content and antioxidant capacity by DPPH free radical scavenging were determined spectrophotometrically. The results showed that the optimal extraction conditions were obtained when adding 1% enzyme α-amylase at 900C in 3.5 hours. The extract of CJR obtained from experiments using enzyme α-amylase showed higher levels of TPC, saponin, antioxidant capacity by DPPH and 0Brix compared to ones without enzymes. The aqueous enzymatic extraction method can be seen as a potential alternative to conventional solvent extraction methods and becomes more popular as an efficient, sustainable, non-toxic and environmentally friendly extraction technique. 1. Introduction Codonopsis javanica (CJ) is a species in the Campanulaceae family, mainly found in mountainous provinces of Northern and Central Vietnam. It is a plant with high medicinal and economic value but more affordable than Korean ginseng. Its roots contain important bioactive compounds such as saponins, phytosteroids, alkaloids and polysaccharides (Chen et al., 2013). It can be used as an ingredient in production of high value products such as functional foods, tea (Thuong et al., 2020), cosmetic material, lotion, etc. (Meng et al., 2022). Proven health benefits of this plant include antioxidant, anti-inflammatory (Do et al., 2022), antibacterial, hypoglycemic, anti-arthritic and anti-diabetic activities, treatment of cough, pulmonary tuberculosis, yellow skin, anaemia and neurasthenia (Dictionary, 2006), reduction of insulin, high blood (Chen et al., 2013) and cancer cells, and body strengthening (Jie et al., 2015) . In 2021, Wu el at studied the active ingredients in ginseng root by water (800C) for 2.2 hours as extraction condition. Figure 1: Dried Codonopsis javanica root in Kon Tum In recent years, most studies have focused on isolating and determining the structure of secondary metabolites in CJR (Phan et al., 2020), polysaccharides purification (Long et al., 2020) or basic extraction process of CJR 553 (Phan et al., 2020; Tri et al., 2020) in Vietnam. There was little scientific information about biological activities of CJR derived from Kon Tum. Food, pharmaceutical and some other related industries have used enzymes in production to increase technological and economic efficiency and improve productivity (Marathe et al., 2017). Enzyme α- amylase helps hydrolyze alpha bonds of polysaccharides such as starch and glycogen, yielding simple substrates such as glucose and maltose. Therefore, in this study, we continued to investigate the water extraction of CJ with the addition of enzyme α- amylase to find the optimal extraction conditions of the water extraction process from roots. Research could be a potential for the development of wellness drinks from CJR grown in Kon Tum, Vietnam. 2. Materials and methods 2.1. Material, chemicals and instrucments Dried Codonopsis javanica root (CJR) (moisture <10%) was purchased from Kon Tum province, Vietnam (Figure 1). Samples were milled, smoothed and stored in dark zip bags (Figure 2). Figure 2: Powder of Codonopsis javanica root Applied chemicals include Enzyme α-amylase from China; Folin (> 95%, Himedia India. DPPH (>96%, China); Methanol, Na2CO3, Vanilin (>99%, Xilong Scientific Co., Ltd) from Germany, distilled water. Experimental tools include Convection dryer (Vietnam), Analytical balance (Model: PA2102, China); Thermostat tank (HH-4, China); UV-VIS spectrophotometer (UV-2602, China). 2.2. Extraction of CJR Take 2g CJR powder, add 40mL water and α-amylase enzyme to the beaker, soak the mixture in a thermostatic bath, then filter with 110 mm diameter filter paper. Finally, the solution was obtained to determine its TPC, antioxidant activity by DPPH, saponin, and 0Brix. The survey parameters include: enzyme α-amylase: 0; 0.25; 0.5; 0.75; 1; 1.25; 1.5% (compared to dried material weight); survey temperature from 80, 85, 90, 95℃ during 2.5h; 3h; 3.5h; 4 h. 2.3. Methodological analysis of saponins, total polyphenols (TPC) and antioxidant activity by DPPH free radical scavenging The total polyphenol content in the sample was determined by the Folin Ciocalteu method with some variations. Put 0.5 ml of sample into a centrifuge tube, then add 2 ml of 10% Folin (v/v), shake well (wait 2-3 minutes). Then add 2 ml of 0.7 M Na2CO3 and continue to wait for 60 min, avoiding direct exposure to lights. TPC was spectrophotometrically measured at 765 nm (Sánchez-Rangel et al., 2013).The DPPH free radical scavenging antioxidant effect was obtained by mixing 3.5 mL of methanol-corrected DPPH with 0.5 mL of the sample solution. After 30 min reaction, determination of antioxidant content was measured at 517 nm in a UV-Vis spectrophotometer (Sharma and Bhat, 2009). Saponin content was determined according to the method described by Hiai et al. (1976). Add 0.5 ml of 8% vanillin to 5 ml of H2SO4 72%. Then add 0.5 ml of the extract, soak the new mixture for about 5 min in cold water (10oC). Then, the mixture was soaked at 60℃ in 15 min for it to rapidly cool again. Saponin content was determined spectrophotometrically at 525 nm. 2.4. Statistical analysis Each experiment was performed 3 times and the collected results were analyzed by descriptive statistics on mean, standard deviation and processed by Excel software. 554 3. Results and discussion 3.1. Effect of enzymes on saponin content, total polyphenol content and antioxidant capacity by DPPH of CJR extract The influence of α-amylase enzyme on biological activities of ginseng root extract is shown in Figure 3. Research conducted to investigate the α-amylase enzyme changed from 0; 0.25; 0.5; 0.75; 1; 1.25; 1.5% (compared to dry material weight) at fixed condition of material/water ratio 1/20 g/mL for 3 hours. According to Figure 3, when changing the enzyme α-amylase, the content of saponin has a slight increase (Figure 3a). After saponin reached the highest value at 1.25% α-amylase enzyme, the saponin content did not change significantly. As for TPC, the graph tends to rise to a peak and then falls (Figure 3b). Specifically, TPC reached the highest value at α- amylase enzyme 0.75%. This could be explained that α-amylase degraded polyphenol into smaller compounds under high enzyme concentrations as stated by Aline et al. (2021). As for the DPPH free radical scavenging antioxidant capacity, it was in the range of 52.3 – 66.9% (Figure 3c). Figure 3: Effect of enzymes on saponin content, total polyphenol content and antioxidant capacity of CJR extract: (a) Saponin content; (b) TPC; (c) DPPH The enzyme α-amylase acted as a catalyst for the hydrolysis reaction. When using the enzyme, the active ingredients of the raw materials were hydrolyzed and released inside the cells (Puri et al., 2012). Thus, in the extraction process with enzymes, biological compounds were generated more. With an aim to increase the amount of active ingredients extracted from the root of the CJ, the survey results showed that when enzyme α-amylase was added to the extraction process, the amount of saponin, TPC and antioxidant capacity by DPPH all gave a higher value than the sample without enzyme α-amylase. The appropriate amount of α- amylase enzyme selected for the following experiments was 1%. 3.2. Effect of extraction time on saponin content, total polyphenol content and antioxidant capacity by DPPH of CJR extract The study carried out at temperature range from 80 to 950C at fixed condition of material/water ratio of 1/20 g/mL during 2.5h; 3h; 3.5h; 4h (Figure 4). When increasing the extraction time from 2.5h to 3.5h, the content of 0 2 4 6 8 10 12 14 0 0.25 0.5 0.75 1 1.25 1.5 Sa po ni n (m g/ g) Enzyme α-amylase (%) 3a 0 1 2 3 4 5 6 7 0 0.25 0.5 0.75 1 1.25 1.5 TP C (m gG A /g ) Enzyme α-amylase (%) 3b 0 10 20 30 40 50 60 70 80 0 0.25 0.5 0.75 1 1.25 1.5 D PP H (% ) Enzyme α-amylase (%) 3c 555 polyphenols and saponins increased slightly. After reaching the highest value at 3.5h, the content of polyphenols and saponins tended to decrease. Increasing the extraction time would facilitate the dissolution of the material and the rapid breakdown of the cell structure. But if the extraction time were too long, it would destroy the unstable active ingredients in the raw materials. Figure 4 showed that the DPPH free radical scavenging ability at 3.5h gave a high value, if the time continued to increase to 4h, the DPPH free radical scavenging ability did not change significantly. To ensure that the content of polyphenols, saponins and high DPPH free radical scavenging values were obtained, the optimal extraction time was selected as 3.5h. The study also gave similar results as the study on the anti-cancer effects of Panax ginseng Berry Polysaccharides (Jie et al., 2015). Figure 4: Effect of extraction time on saponin content, total polyphenol content and DPPH antioxidant capacity of the extracts: (a) Saponin content; (b) TPC; (c) DPPH 3.3. Effect of extraction temperature on saponin content, total polyphenol content and DPPH antioxidant capacity of the extracts Experiments to investigate the effect of extraction temperature on biological activities of extracts were conducted at the following conditions: ratio 1/20g/mL, 1% enzyme, extraction time 3.5h. Figure 5 showed the change of the survey parameters when changing the extraction temperature from 80, 85, 90, 950C. Specifically, when increasing the soaking temperature, the TPC, saponin values and DPPH free radical scavenging ability also increased. All three analytical parameters reached the maximum value at the temperature of 900C and then decreased accordingly. The right soaking temperature would help active ingredients in the raw materials be released to the outside environment. But if the temperature were too high, it denatured those biologically active substances. The research results were higher than those reported by Lee et al. (2008) when performing saponin extraction from Korean red ginseng (800C). This difference could result from different sources of local materials and survey methods. 0 2 4 6 8 10 12 14 2.5h 3h 3.5h 4h Sa po ni n (m g/ g) Extraction time 4a 0 1 2 3 4 5 6 7 2.5h 3h 3.5h 4h TP C (m gG A /g ) Extraction time 4b 52 53 54 55 56 57 58 59 60 61 2.5h 3h 3.5h 4h D PP H (% ) Extraction time 4c 556 Figure 5: Effect of extraction temperature on saponin content, total polyphenol content and DPPH antioxidant capacity of the extracts: (a) Saponin content; (b) TPC; (c) DPPH Table 1 showed that the CJR extract obtained pleasant characteristics with light yellow, aromatic, sweet and sour in sense. Table 1: Biological properties of CJR extract with 1% enzyme α-amylase treatment № Target Result 1 Sense gold; light aroma; sweet, sour 2 0Brix 4.2% 3 pH 5.5 4 Saponin 11.67 mg/g 5 DPPH free radical scavenging ability 79% 6 TPC 6.48 mgGA/g 7 Moisture <10% 4. Conclusion Codonopsis javanica root from Kon Tum, Vietnam has long been regarded as a valuable source of biologically active ingredients widely used in food, cosmetic, oriental and modern medicine. The research results showed that the optimal conditions for extraction of CJR by immersion in thermostatic bath were found when using 1% enzyme α-amylase at 900C, 3.5h and 1/20 g/mL. The total phenol content of the aqueous extract was 6.48 mgGA/g, saponin content 11.67 mg/g and DPPH free radical scavenging ability 79%. Codonopsis javanica root extracted with enzyme had 0Brix value (4.2%) higher than without-enzyme treatment (2.2%). Acknowledgments We are grateful to Van Lang University, Vietnam for the financial support for this study. 0 2 4 6 8 10 12 14 80 85 90 95 Sa po ni n (m g/ g) Extraction temperature (0C) 5a 0 1 2 3 4 5 6 7 80 85 90 95 TP C (m gG A /g ) Extraction temperature (0C) 5b 0 10 20 30 40 50 60 70 80 90 80 85 90 95 D PP H (% ) Extraction temperature (0C) 5c 557 References Cascaes-Teles A.S., Chavez D.W.H., Coelho M.A.Z., Rosenthal A., Gottschalk L.M.F., Tonon R.V., 2021, Combination of enzyme-assisted extraction and high hydrostatic pressure for phenolic compounds recovery from grape pomace, Journal of Food Engineering 288, 110128. Chen K.N., Peng W.H., Hou C.W., Chen C.Y., Chen H.H., Kuo C.H., Korivi M., 2013, Codonopsis Javanica Root Extracts Attenuate Hyperinsulinemia and Lipid Peroxidation in Fructose-Fed Insulin Resistant Rats, Journal of Food and Drug Analysis 21 (4), 347–55. 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