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Vietnam Journal of Science and Technology 58 (6A) (2020) 91-101 doi:10.15625/2525-2518/58/6A/15473 EXTRACTION OF TRITERPENOIDS FROM THE VIETNAMESE RED GANODERMA LUCIDUM BY ULTRASOUND-ASSISTED EXTRACTION METHOD AND ANTI-OXIDANT ACTIVITY OF EXTRACT Tran Do Dat1, Nguyen Tan Tai2, Phan Le Thao My1, Ngo Thi Thuy Linh1, Nguyen Thi Kim Ngan1, Vuong Hoai Thanh2, Nguyen Duc Viet2, Hoang Minh Nam2, 3, Mai Thanh Phong2, 3, Nguyen Huu Hieu1, 2, 3, * 1 VNU-HCMC Key Laboratory of Chemical Engineering and Petroleum Processing (CEPP Lab), Viet Nam, 70000 2 Faculty of Chemical Engineering, Viet Nam, 70000 Ho Chi Minh City University of Technology, 268 Ly Thuong Kiet Street, Ward 14, District 10, Ho Chi Minh City, Viet Nam 3 Vietnam National University Ho Chi Minh City, Linh Trung Ward, Thu Duc District, Ho Chi Minh City, Viet Nam * Email: nhhieubk@hcmut.edu.vn Received: 9 September 2020; Accepted for publication: 27 January 2021 Abstract. An ecofriendly ultrasound – assisted extraction (UAE) method was developed for the triterpenoids extraction from Vietnamese red Ganoderma lucidum (G. lucidum). The appropriate kind of solvent for extraction procedure was selected as ethanol based on the yield of triterpenoids obtained, followed by the investigation of four single factors including solvent-tomaterial ratio, extraction temperature, extraction time, and ultrasonic power. According to the result of single factor experiments, optimal extraction conditions were determined with solventto-material ratio of 30:1 mL/g, extraction temperature of 55 oC, extraction time of 60 min, and ultrasonic power of 480 W. Under these conditions, UAE produced a highest triterpenoids yield of 7.479 0.033 mg/g by using ultraviolet-visible spectroscopy (UV-Vis) analysis. Extract obtained from UAE method showed mild antioxidant activity compared to that of ascorbic acid with the half-maximal inhibitory concentration values were of 29.778 and 6.714 µg/mL, respectively. As a result, triterpenoids extracted from G. lucidum could be considered as a promising antioxidant agent. Keywords: Ganoderma lucidum, ultrasound – assisted extraction, triterpenoids. Classification numers: 1, 3. 1. INTRODUCTION Belonging to the Ganodermataceae family, Ganoderma lucidum (G. lucidum) is one of functional foods used to boost health and longevity, especially in China [1]. Various components from G. lucidum including the fruiting body, spores, and mycelia have a long history of use for Tran Do Dat, et al. preventing and treating numerous diseases such as hepatitis, bronchitis, nephritis, hypertension, and cancer [2 - 5]. It is reported that G. lucidum contains diverse phytochemicals including polysaccharides, triterpenoids, oils, sterols, organic germanium, and inorganic ions [6]. Triterpenoids with over 400 compounds have been found and many more still being discovered, are regarded as one of the crucial bioactive components exhibiting anticancer, antiinflammatory, antioxidant, anti-proliferation, antiangiogenesis, and anti-lymphatic effects [7 11]. Therefore, effective extraction methods for triterpenoids from G. lucidum needed to be developed. As new-type extraction technique, ultrasound – assisted extraction method (UAE) has many advantages compared to conventional techniques including shorter extraction time, higher extraction rate, reduction in the amount of solvent used, and increase in purity of product obtained. In addition, the easy maintenance of the equipment area enhances the wide applicability of UAE method [12 - 14]. The principle of UAE is based on the ability of continuous compression generation and rarefaction with the assistance of an extraction solvent leading to formation of micro-bubbles. As a result, substances could be released effectively from plant materials [12,15]. However, extraction efficiency is affected by many process variables such as solvent type, solvent-to-material ratio, extraction time, and extraction temperature [16]. The objective of the work was to determine the applicability of UAE method for a reliable and reproducible extraction process of triterpenoids from G. lucidum as well as evaluate the antioxidant activity of the extract obtained in comparison with ascorbic acid. 2. MATERIALS AND METHODS 2.1. Materials and chemicals The fruiting body of Vietnamese G. lucidum was provided by National Institute of Medicinal Materials with the moisture value of materials at 12 %, Viet Nam. 99.5 % ethanol (C2H5OH) was purchased from ChemSol, Viet Nam. 99.0 % methanol (CH3OH), 70.0 - 72.0 % perchloric acid (HClO4), acetic acid (CH3COOH), ascorbic acid, vanillin, and ursolic acid were purchased from Xylong, China. Double-distilled water was used in all experiments. All chemicals were used as received without further purification. 2.2. Selection of appropriate kind of solvent for extraction procedure The influences of different kinds of solvents including ethanol, water, hexane, methanol, and ethyl acetate on the yield of triterpenoids were examined while extraction conditions were fixed as follows: solvent-to-material ratio of 30:1 mL/g, extraction time of 60 min, extraction temperature of 55 , and ultrasonic power of 480 W. 2.3. Experiment procedure After being dried at 50 oC to remove moisture, 2.00 g of dried powder of Vietnamese G. lucidum was accurately weighed and dispersed in the appropriate kind of extraction solvent with the assistance of ultrasound wave. Then the samples were filtered and concentrated under reduced pressure using the vacuum evaporation to obtain crude extracts. 2.4. Determination of the yield of triterpenoids 92 Extraction of triterpenoids from the Vietnamese red Ganoderma lucidum … The yield of triterpenoids was evaluated based on previous study [17]. Ursolic acid was used as a standard and an adequate range of concentrations including 0.02, 0.03, 0.04, 0.05, and 0.06 mg/mL in ethyl acetate was used to create a calibration curve. 1.0 mL of perchloric acid solution and 0.4 mL of 5 % vanillin-glacial acetic acid reagent were added to the extract sample. Reaction mixture was further heated for 60 oC for 15 min and then cooled in an ice-water bath. Thereafter 5.0 mL of glacial acetic acid solution was mixed with it and the absorbance of the sample solutions was measured at 548 nm using ultravioletvisible spectroscopy analysis against a blank (ethyl acetate). All of determinations were made in triplicate. The yield of triterpenoids (%) was calculated by the following equation: ( ) (1) where Y is the yield of triterpenoids (%); C is the concentration of triterpenoids obtained from the calibrated regression equation (mg/mL); V is the volume of triterpenoids solution (mL); n is the dilution factor; and m is the dried sample mass (g). 2.5. Single factor experimental design The effects of factors on the yield of triterpenoids, including solvent-to-material ratio (20:1, 25:1, 30:1, 35:1, and 40:1 mL/g), extraction time (30, 60, 90, 120, and 150 min), ultrasonic power (240, 360, 480, 600, and 720 W), and extraction temperature (45, 50, 55, 60, and 65 oC) were determined based on the simultanous influences of the factors to select main factors. Throughout the single-factor experiments, one variable was changed while the other variables were kept constant. 2.6. Anti – oxidant activity investigation of the extract The scavenging of DPPH radicals was assayed according to previous study [18]. 4.0 mL of sample extract was added an equal volume of 6.0 mL of 1 mM DPPH solution with methanol. The mixture was then mixed well and allowed in the dark for 30 min at room temperature before the absorbance of the mixture was read at 517 nm. The anti-oxidation activity is proportional to the disappearance of DPPH. The ascorbic acid was used as a positive control sample in DPPH anti-oxidation activity test. A different concentration of ascorbic acid (1.0, 2.0, 4.0, 6.0, 8.0, and 10.0 µg/mL) was prepared and the procedure was carried out in the same manner as for the sample extract. All the experiments were conducted in triplicate. 2.7. Statistical analysis The one-way analysis of variance (ANOVA) test with least significant difference (LSD) was used to statistically investigate the average yield of triterpenoids. The software package Statgraphics Centurion 18 (Statgraphics Technologies, Inc., Warrenton, VA, USA) was employed for the statistical data evaluation. The results were expressed as mean standard deviation (SD) (n = 3). The p values less than 0.05 or less than 0.01 are considered significant or highly significant, respectively. All graphs were plotted using OriginPro 8.5.1 (OriginLab Corporation, Northampton, MA, USA). 93 Tran Do Dat, et al. 3. RESULTS AND DISCUSSION 3.1. Selection of appropriate kind of solvent for triterpenoids extraction procedure Figure 1 shows the effect of different kinds of solvents including n-hexane, ethyl acetate, water, ethanol, and methanol on the yield of triterpenoids under extraction conditions including solvent-to-material ratio of 35:1 mL/g, extraction temperature of 55 oC, extraction time of 60 min, and ultrasound power of 480 W. Figure 1. Effect of different kinds of solvents on the yield of triterpenoids. Dissimilar letters in the same column indicate significantly different at p < 0.05 using one-way ANOVA. As can be seen from Figure 1, the highest yields of triterpenoids could be obtained when methanol and ethanol were used as solvents, while the extraction yield obtained using water, ethyl acetate, and n-hexane were 5.3568, 4.7730, and 2.3399 mg/g, respectively. This result could be explained due to the fact that medium polar solvents could facilitate extraction efficiency [19]. However, methanol is regarded as a highly toxic solvent while ethanol is a safe solvent with good yield of triterpenoids. Therefore, ethanol was selected as the most appropriate solvent used for the extraction process. It is reported that ethanol was widely used as an efficient solvent for triterpenoid extraction [20, 21]. 3.2. Effect of single factors 3.2.1. Extraction temperature Figure 2 illustrates the effect of extraction temperature on the yield of triterpenoids and extraction conditions were set as solvent-to-material ratio of 30:1 mL/g, extraction time of 60 min, ultrasound power of 480 W with using ethanol as extraction solvent. As shown in Figure 3, the yield of triterpenoids increased from 5.00 0.069 to 7.480 ± 0.234 mg/g when extraction 94 Extraction of triterpenoids from the Vietnamese red Ganoderma lucidum … Yield of triterpenoids (mg/g) temperature was increased from 45 to 55 oC. After that, the yield of triterpenoids tended to decrease although extraction time continued to rise. It was expected that enhancement of extraction temperature would lead to higher extraction efficiency due to improvement in desorption of analytes from the active sites in the matrix, solvent capacity of solubilizing analyte, and sample penetration [22, 23]. When the extraction temperature of above 55 was processed, high temperature could cause five – ring structures of triterpenoid molecules destruction based on reported studies [24, 25]. A maximum yield of triterpenoids was achieved when using an extraction temperature of 55 oC and this value was chosen for subsequent experiments. Extraction temperature (oC) Figure 2. Effect of extraction temperature on the yield of triterpenoids. Dissimilar letters in the same column indicate significantly different at p < 0.05 using one-way ANOVA. Yield of triterpenoids (mg/g) 3.2.2. Extraction time Extraction time (min) Figure 3. Effect of extraction time on the yield of triterpenoids. Dissimilar letters in the same column indicate significantly different at p < 0.05 using one-way ANOVA. 95 Tran Do Dat, et al. Figure 3 demonstrates the effect of extraction time on the yield of triterpenoids when the experimental conditions including solvent-to-material ratio of 30 mL/g, extraction temperature of 55 oC, extraction time of 60 min, and ultrasound power of 480 W. The result indicated that with increasing extraction time until 60 min, the yield of triterpenoids increases sharply and peaked at 7.472 0.024 mg/g, after which a slight reduction is observed. During the ultrasonic process, it is reported that with longer extraction time as ultrasound wave could facilitate the equilibrium establishment for the dissolution of triterpenoids between the plant cell wall and extraction solvent in a short time [23, 26]. However, prolonged extraction time could exert an adverse impact on the yield of triterpenoids as keeping at high temperature for a long time period results in decomposition of triterpenoids [27]. This result is suitable with previous research [25, 28]. Consequently, 60 min was chosen for further experiments. 3.2.3. Ultrasonic power Yield of triterpenoids (mg/g) As shown in the Figure 4, the effect of ultrasonic power on the yield of triterpenoids was investigated while the other extraction parameters were set as follows: solvent-to-material ratio of 30 mL/g, extraction time of 60 min, extraction temperature of 55 oC. When ultrasonic power was varied from 200 to 500 W, the yield of triterpenoids was increased considerably and obtained its maximum value of 7.479 0.033 mg/g at ultrasound power of 480 W, then sharply reduced. It could be explained that greater ultrasound power would accelerate formation and collapses of burble at the plant surface interface, thus drastically destroy cell walls and release components within the cells. Nevertheless, thermal degradation resulted from local heating effect induced by sonication. This result is in agreement with previous research [25]. Hence, 480 W was selected for the next study. Ultrasonic power (W) Figure 4. Effect of ultrasonic power on the yield of triterpenoids. Dissimilar letters in the same column indicate significantly different at p < 0.05 using one-way ANOVA. 3.2.4. Solvent-to-material ratio 96 Extraction of triterpenoids from the Vietnamese red Ganoderma lucidum … Figure 5 presents the effect of solvent-to-material ratio on the yield of triterpenoids while triterpenoids was extracted using ethanol with extraction temperature of 55 , extraction time of 60 min, and ultrasound power of 480 W. It is clearly that the yield of triterpenoids rose dramatically when the solvent-to-material ratio increased. The highest yield of triterpenoids obtained the value of 7.475 0.045 mg/g at the solvent-to-material ratio of 30:1 mL/g. It is stated that the sufficient volume of solvent must be used for the immersion of entire sample [22]. Generally, extraction yield improvement could be acquired when there is an increment of solvent-to-material ratio as substances dissolve more effectively [23]. Nevertheless, large solvent-to-material ratio would lead to a decrease in the yield of triterpenoids due to the negative influence on the cell wall material break and mass transfer kinetics. This result is consistent with previous studies [24]. As a result, the solvent-to-material ratio of 30:1 mL/g was used to conduct the following experiments. 7.475 ± 0.045 c 6.495± 0.044 6.069 ± b 0.109 4.980 ± 0.069 6.480 ± 0.066 d d a Figure 5. Effect of solvent-to-material ratio on the yield of triterpenoids. Dissimilar letters in the same column indicate significantly different at p < 0.05 using one-way ANOVA. The influence of four factors on triterpenoid content was determined by ANOVA with the use of LSD measuring the different meaning (p < 0.05) in order to compare the average difference between four variables. Table 1 demonstrates the effects of four operating parameters on the triterpenoid content. Fromt the result, four operating parameters namely extraction time, extraction power, extraction temperature and solvent-to-material ratio were reported to illustrate the same level of influence with solvent-to-material ratio. Table 1. The effects of four operating parameters on the triterpenoid content. Operating parameters Count Mean Homogeneous Groups Extraction time 3 7.472 X Ultrasonic power 3 7.479 X X Extraction temperature 3 7.48 X Solvent-to-material ratio 3 7.475 X 97 Tran Do Dat, et al. 3.3. Antioxidant activity investigation of the extract Figure 6 presents the anti-oxidant activity of different concentrations of ascorbic acid (a) and the extract (b) in DPPH scavenging assay. As can be seen from Figure 6, the half-maximal inhibitory concentration (IC50) value of the extract and ascorbic acid was 29.778 and 6.714 µg/mL, respectively. Thus, the IC50 value of the extract was 4.44 times higher than that of ascorbic acid. In preliminary studies, the anti-oxidant activities of Vietnamese G.lucidum have been in association with the presence of phenolic, triterpenoids, and polysaccharide compounds [29,30]. This could be elucidated by several reasons. Firstly, the antioxidant activities of the extract have been revealed by four isolated polysaccharides with different compositions in its structures [30]. In the second explanation, it might be due to reduction of molecular weight by ultrasound waves. In addition, it was reported that the break of polysaccharides into monosaccharides would horn the ability to scavenge DPPH- radicals according to previous studies [31]. (a) (b) Figure 6. Anti-oxidation activity of different concentrations of ascorbic acid (a) and the extract (b) in DPPH scavenging assay. 4. CONCLUSIONS In this study, triterpenoids were extracted from Vietnamese red G. lucidum by using UAE method. The highest yield of triterpenoids obtained under the optimal extraction conditions including solvent-to-material ratio of 30:1 mL/g, extraction temperature of 55 , extraction time of 60 min, and ultrasonic power of 480 W was reported to be of 7.479 0.033 mg/g based on ultraviolet-visible spectroscopy analysis. The extract obtained from UAE method showed low antioxidant activity in comparison with that of ascorbic acid, with IC50 values were of 29.778 and 6.714 µg/mL, respectively. Therefore, UAE could be useful for extraction of triterpenoids from Vietnamese red G. lucidum and further studies related to the application of this modern extraction technique need to be conducted. Acknowledgments. This research is funded by Vietnam National University Ho Chi Minh City (VNUHCM) under grant number B2019-20-02. We acknowledge the support from Ho Chi Minh City University of Technology (HCMUT), VNU-HCM for this study. 98 Extraction of triterpenoids from the Vietnamese red Ganoderma lucidum … CRediT authorship contribution statement. TDD: Methodology, Investigation, Writing - original draft. NTT: Methodology, Investigation, Writing - original draft. PLTM: Investigation, Writing - review & editing. NTTL: Conceptualization, Methodology, Investigation, Writing - original draft. NTKN: Investigation, Writing - review & editing. VHT: Writing - review & editing. NDV: Validation, Formal analysis, Data curation, Supervision. 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