Supercritical Fluid Extraction of Polyphenols from Vietnamese Callisia fragrans Leaves and Antioxidant Activity of the Extract

VNU-HCMC Key Laboratory of Chemical Engineering and Petroleum Processing (CEPP Lab), Ho Chi Minh City University of Technology, 268 Ly )uong Kiet Street, Ward 14, District 10, Ho Chi Minh, Vietnam Faculty of Chemical Engineering, Ho Chi Minh City University of Technology, 268 Ly )uong Kiet Street, Ward 14, District 10, Ho Chi Minh, Vietnam Hong Bang International University, 215 Dien Bien Phu Street, Ward 15, Binh )anh District, Ho Chi Minh, Vietnam Vietnam National University, Linh Trung Ward, )u Duc District, Ho Chi Minh, Vietnam


Introduction
Over 500 species belonging to Commelinaceae family, especially Vietnamese Callisia fragrans (C. fragrans), have been widely grown in eastern Australia to have their leaves and stems harvested [1]. Containing a great variety of bioactive components such as flavonoids, phytosteroids, vitamins, and microelements, this plant has been well known for many pharmaceutical uses including anti-inflammation, pain killers, burn treatment, and cardiovascular drugs [2,3].
Polyphenols, known as secondary metabolites, present commonly in all higher plants with more than 8,000 phenolic structures that were identified and classified into 10 classes in association with its structures [4]. ese compounds consist of flavonoids and nonflavonoids. It is reported that there are more than 4,000 flavonoid structures of which 143 are proanthocyanidins that have been found [5]. In terms of nonflavonoids, phenolic acids play a pivotal role and have been seen as derivatives of benzoic acids comprising gallic acid, protocatechuic acid, and derivatives of cinnamic acid [6]. In addition, polyphenols have been considered as an antioxidant agent and an anticancer component owing to biological characterizations for the activation of a variety of enzymes and cell receptors [7]. e polyphenols have been widely applied for sunburn prevention because their aromatic-ring structures could affect the absorbance of the UV radiation ranging from 280 to 315 nm [8].
e solid-liquid extraction method has been divided into two types encompassing conventional and modern technologies. Conventional extraction techniques such as maceration, percolation, and decoction have long been applied in the fields of pharmacy and food industry [9]. Since the fundamental principles of the Soxhlet extraction (SE) method rely on exhaustive extraction leading to higher yields, this technique has been usually utilized in the laboratory due to its limitations of raw materials and well employed mainly for vegetable oil extraction compared to other traditional techniques such as maceration [10]. With the advantage of the amount of solvent reduction, this method could take long extraction time, contribute to pollution problems because of flammable and hazardous solvent consumption, and require strictly high-purity solvents [11]. Because of the development and enhancement of green extraction technologies, the application of ultrasound, microwave, enzyme, and pressurized liquid has been widely used in many parts of the world [12]. Among these methods, the employment of supercritical fluid, especially supercritical carbon dioxide (SC-CO 2 ) extraction method, has been globally investigated to extract natural products from plants based on its distinct capacities [13]. For the utilization of supercritical fluid technology, it has been gained the interest of food and pharmaceutical industries due to the prevention of thermal degradation of target compounds [14]. For instance, the industrial levels of SC-CO 2 extraction have been started since the 1970s for decaffeination of tea and coffee [15]. In contrast with polar phenolic structures, the nonpolar carbon dioxide (CO 2 ) compound may modify its polarity by the integration of other solvents called cosolvent or modifier such as ethanol to horn the selectivity and extraction yield of final products [16]. According to preliminary studies, temperature and pressure play a pivotal role in the extraction process using SC-CO 2 [10]. Besides, cosolvent concentration and CO 2 flow rate have been reported as positive factors, having great impacts on extraction yields [17].
In this study, from Vietnamese C. fragrans, based on total polyphenol content (TPC) determination and antioxidant activity investigation of the extracts obtained, the effect of single operational parameters including ethanol concentration, CO 2 flow rate, extraction temperature, and pressure on extraction process was evaluated by utilizing ethanol as a cosolvent for SC-CO 2 extraction method. In addition, SE method was carried out in comparison with the SC-CO 2 extraction method to determine the efficient modern extraction method for polyphenols.

Materials and Reagents.
e leaves of Vietnamese C. fragrans were collected from Hoc Mon District, Ho Chi Minh City, Vietnam. After harvesting, the leaves were cleaned and dipped in 70.0% ethanol before being air-dried for 1 h. en the leaves were stored in dark bags to prevent polyphenol degradation due to sunlight and transferred immediately to the laboratory in 1 to 1.5 h. By using a dry air oven, the leaves were dried at a temperature of 70°C for 3-4 h until a constant weight was obtained. Before extraction, the leaves were ground to a fine powder with a commercialgrade blender and stored at −20°C for further analysis.

2.2.1.
e Removal of Nonpolar Organic Molecules. 500.0 g of leaf powder of Vietnamese C. fragrans leaves was extracted with n-hexane by maceration for 14 days (1.5 L of n-hexane solvent, each day) at room temperature to remove nonpolar impurities. After that, the leaf powder was shadedried at room temperature for 1 day and marked as LH.

SE Method.
To compare the extraction efficiency, 15.0 g of the leaf powder LH was extracted by the SE method with 350 mL of ethyl acetate, extraction temperature of 80-85°C, and extraction time of 6 h. e collected extracts were concentrated under reduced pressure to partly remove solvents by using a vacuum evaporator at a temperature of 35°C to obtain crude extract marked as CLE.

Effect of Single Factors on TPC.
In this study, the influences of four single operational parameters including ethanol concentration, CO 2 flow rate, extraction temperature, and pressure were determined according to the TPC and antioxidant activity of the extracts obtained from singlefactor experiments.

TPC Determination.
TPC was determined based on the Folin-Ciocalteu method [12]. Folin-Ciocalteu's reagent was used to oxidize the samples, and the absorbance measurement was read at 760 nm after 60 min. Gallic acid was used as a standard to create calibration, and TPC was expressed as gallic acid equivalent per gram of dry weight of the Vietnamese C. fragrans extract.

e Antioxidant Activity Investigation of the Extracts.
e extracts obtained from each single-factor experiment were tested for antioxidant activity by ABTS (2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium) radical cation decolorization assay [18]. e ABTS radical cation (ABTS+) could be generated by mixing the 2.45 mM potassium persulfate solution and 7 mM ABTS solution with the ratio of 1 : 1. is reaction mixture was then kept in the dark at room temperature for 12-16 h before use. e absorbance of the sample was read at 734 nm. e Trolox was used as a standard in the antioxidant activity experiments to generate the calibration curve, and the antioxidant activity of the extract was expressed as μmol TE (Trolox equivalent) per gram of dry weight of the Vietnamese C. fragrans extract. e percentage ABTS inhibition was calculated using the following equation: where A 1 is the absorbance of the sample reaction solution and A 0 is the absorbance of the free radical solution. IC 50 value was the inhibitory concentration of antioxidant needed to decrease the initial radical by 50%. e IC 50 values were obtained from the standard curve, and a lower IC 50 value represents a stronger ABTS scavenging capacity.

Effect of Single Factors on the TPC
3.1.1. Ethanol Concentration. Figure 1 indicates the effect of ethanol concentration on the TPC and antioxidant activity of the extracts. Experiments were conducted under extraction conditions including extraction temperature of 50°C, extraction time of 60 min, CO 2 flow rate of 14 g/min, and pressure of 200 bar. As can be seen from Figure 1, TPC increased from 35.54 mg GAE/g to 37.56 mg GAE/g with an increase in ethanol concentration between 10 and 14% and went down to 30.58 mg GAE/g when ethanol concentration surpassed over 14%. According to preliminary studies, a nonpolar CO 2 compound could be modified with a polar solvent such as methanol or ethanol to horn extraction yield. Although methanol might be the most efficient solvent used, it is more toxic compared to ethanol [19]. It was also reported that using ethanol as a binary solvent could lead to an increase in the TPC because of the alteration of its polarity. Besides, the cell permeability would be enhanced by this solvent as ethanol could have an impact on the phospholipid bilayer of the cell membrane [16]. Additionally, kinds of polyphenols extracted by ethanol, as is known from several studies, are strongly dependent on ethanol concentration [20][21][22]. e antioxidant activity of the extracts experienced an upward trend and reached its maximum value of 181.58 μM TE/g at an ethanol concentration of 14%. However, as the ethanol concentration continued to rise above 14%, antioxidant activity fell down rapidly. is could be articulated by the strong relationship between ethanol concentration and free radical properties of the crude extract due to the hydroxyl groups of polyphenols [23]. In accordance with other studies, it is revealed that the antioxidant activity of polyphenols has been strongly relevant to its structures [4]. Hence, higher ethanol concentration could be used to improve the effective dissolution of polar polyphenols. However, too high ethanol concentration may lead to dissolving byproduct compounds, resulting in the decrease of antioxidant capacities. Ethanol concentration changes cause solvent physical properties such as dynamic viscosity, density, and dielectric constant modification, influencing the phenolic compound extraction based on previous studies [24]. erefore, 14% was chosen as an optimal ethanol concentration for further experiments. Figure 2 presents the effect of the CO 2 flow rate on the TPC and antioxidant activity of the extract. To investigate the impact of the CO 2 flow rate, polyphenolics were extracted using 10% ethanol concentration at 50°C for 120 min with pressure of 200 bar. From Figure 2, it can be seen that TPC increased while rising CO 2 flow rate and gained the highest value of 65.58 mg GAE/g at 20 g/min. In terms of antioxidant properties, the antioxidant activity of the extracts grew up gradually and peaked at 192.58 μM TE/g as the highest point with CO 2 flow rate value of 20 g/min. An increase in TPC could be elucidated by an elevation in the quantities of ethanol and CO 2 compounds presenting in samples, leading to strong interactions between solvents and target compounds. Additionally, adequate extraction time would positively contribute to the growth of TPC because it is relevant to mass transfer kinetics [17,25]. e results would be in agreement with reported research [26]. Hence, the CO 2 flow rate of 20 g/min was selected as the optimum. Figure 3 shows the effect of temperature on the TPC and antioxidant activity of the extract. Polyphenols were extracted under extraction conditions including CO 2 flow rate of 14 g/min, extraction time of 120 min, ethanol concentration of 10%, and pressure of 200 bar.

Extraction Temperature.
It can be seen that increasing the extraction temperature from 40 to 45°C generally increases TPC and antioxidant activity of the extracts with the maximum values of 43.95 mg GAE/g and 180.5 μM TE/g, respectively. When the extraction temperature was over 45°C, a slight decrease in the TPC and antioxidant activity of the extracts could be observed.
is phenomenon could be explained due to the fact that as increasing temperature until 45°C, the TPC elevated because of an increase in diffusion and desorption [19]. Moreover, raising temperature could be ascribed to some aspects such as permeability of cell walls, solubility of targeted Journal of Chemistry compounds, and mass transfer phenomena [16]. It is cited that polyphenols such as anthocyanins are thermally unstable and easily hydrolyzed or oxidized. Hence, it could rapidly degrade by high temperature. In addition, the process of polyphenol hydroxylation and oxidization might induce a decrease in its antioxidant activity of the extracts [27]. e enhancement of temperature has directly exerted a detrimental impact on reducing the density of the modified supercritical CO 2 , contributing to a decrease of the solubility [28]. is result could be found familiar with other research [29,30]. us, the extraction temperature of 45°C was selected to employ for further research. Figure 4 describes the effect of pressure on the TPC and antioxidant activity of the extract. e influence of pressure on the TPC and antioxidant activity was studied at 100, 150, 200, and 250 bars with CO 2 flow rate of 14 g/min, extraction time of 60 min, ethanol concentration of 10%, and extraction temperature of 50°C. As shown in Figure 4, the TPC and antioxidant activity of the extracts were enhanced and obtained its highest content of 33.55 mg GAE/g and 105.41 μM TE/g, respectively, upon increasing pressure to 200 bar, after which a gradual decline in the TPC and antioxidant activity of the extracts could be observed. It is reported that the main important mechanism could be used to explain for this would be the fluid density, which changes the solubility of the solute [31]. Although the density boosts the solvating power of CO 2 while increasing pressure, higher pressure could lead to the low mass transfer kinetics due to the low dispersion coefficient of supercritical CO 2 as falling diffusivity, negative influence on the highly porous extraction bed, and diminishing in the interaction between solute and solvent.

Pressure.
us, extraction efficiency and antioxidant activity decreased sharply [28,32]. From the previous results, this study might be in consonance with other research [30,33]. As a result, 200 bar would be set as an optimal pressure.  It is cited that among these conventional techniques, SE has been widely used for polyphenol extraction because employing higher temperature leads to higher mass transfer kinetics as well as continuous change in the transfer equilibrium by the new contact between fresh solvents and solid particles [10]. However, the method could have several restrictions such as long time extraction and volatile as well as flammable organic solvents consumption. ese disadvantages might be altered by SC-CO 2 , which could generate an improvement in extraction yields and antioxidant activity [11]. In application scale, with limitation on the amount of materials used, SE could not be scalable compared to SC-CO 2 as SC-CO 2 may be suitable for the large quantity of mass production without contaminating products with solvent residues [34]. is result is consistent with reported studies [3,35,36].

Conclusions
From Vietnamese C. Fragrans, based on the highest TPC and antioxidant activity of the extract obtained from each singlefactor experiment, the extraction conditions determined with the application of SC-CO 2 method using ethanol as a cosolvent were ethanol concentration of 14%, CO 2 flow rate of 20 g/min, extraction temperature of 45°C, and pressure of 200 bar. Under these conditions, the extract was compared with that obtained under the SE method in terms of TPC and antioxidant activity. e results showed that the TPC and the antioxidant activity with IC 50 values of the extract obtained using SC-CO 2 method were of 87.42 ± 1.33 mg/g and 243.83 ± 5.30 μM TE/g, respectively, while that obtained using the SE method were of 85.34 ± 4.27 mg/g and 236.33 ± 7.66 μM TE/g, respectively. In terms of polyphenol composition determination by HPLC method for isolation and purification of polyphenol compounds, further research would be conducted in the next study. erefore, SC-CO 2 method could be considered as a potential efficient extraction method in comparison with the conventional SE method.

Data Availability
e data used to support the findings of this study are included within the article.

Conflicts of Interest
e authors declare that they have no conflicts of interest.