传统溶剂提取法和超临界流体萃取法对钟花蒲桃次生代谢产物的回收率的比较研究

A comparative study of conventional and supercritical fluid extraction methods for the recovery of secondary metabolites from Syzygium campanulatum Korth # # 传统溶剂提取法和超临界流体萃取法对钟花蒲桃次生代谢产物的回收率的比较研究 1 first-author 1 1 2 2 1 1 corresp ‡Corresponding Author ‡Corresponding Author †E-mail:ismailzhari@gmail.com †E-mail:ismailzhari@gmail.com ismailzhari@gmail.com 3 Department of Pharmaceutical Chemistry, School of Pharmaceutical Sciences, Universiti Sains Malaysia, 11800 Minden, Penang, Malaysia Department of Pharmaceutical Chemistry, School of Pharmaceutical Sciences, Universiti Sains Malaysia, 11800 Minden, Penang, Malaysia Center of Lipids Engineering and Applied Research (CLEAR), Ibnu Sina Institute for Scientific & Industrial Research (Ibnu Sina ISIR), Universiti Teknologi Malaysia, 81310 Johor Bahru, Johor, Malaysia Center of Lipids Engineering and Applied Research (CLEAR), Ibnu Sina Institute for Scientific & Industrial Research (Ibnu Sina ISIR), Universiti Teknologi Malaysia, 81310 Johor Bahru, Johor, Malaysia EMAN Research and Testing Laboratory, School of Pharmaceutical Sciences, Universiti Sains Malaysia, 11800 Minden, Penang, Malaysia EMAN Research and Testing Laboratory, School of Pharmaceutical Sciences, Universiti Sains Malaysia, 11800 Minden, Penang, Malaysia Syzygium campanulatum Korth is a plant, which is a rich source of secondary metabolites (especially flavanones, chalcone, and triterpenoids). In our present study, three conventional solvent extraction (CSE) techniques and supercritical fluid extraction (SFE) techniques were performed to achieve a maximum recovery of two flavanones, chalcone, and two triterpenoids from S. campanulatum leaves. Furthermore, a Box-Behnken design was constructed for the SFE technique using pressure, temperature, and particle size as independent variables, and yields of crude extract, individual and total secondary metabolites as the dependent variables. In the CSE procedure, twenty extracts were produced using ten different solvents and three techniques (maceration, soxhletion, and reflux). An enriched extract of five secondary metabolites was collected using n -hexane:methanol (1:1) soxhletion. Using food-grade ethanol as a modifier, the SFE methods produced a higher recovery (25.5%‒84.9%) of selected secondary metabolites as compared to the CSE techniques (0.92%‒66.00%). 目的 2 寻求一种最佳的钟花蒲桃次生代谢产物的萃取方法，以实现在工业上的应用。 创新点 2 全面深入地比较传统溶剂提取法和超临界流体萃取法对钟花蒲桃的主要次生代谢产物的回收率。 方法 2 在超临界流体萃取法中，应用Box-Behnken设计，以压力、温度和萃取粒径大小为自变量，以粗提取物、个别的和总的次生代谢产物的百分比产量为因变量。另外，还研究了调节剂乙醇对产量的影响。在传统溶剂提取中，结合十种不同的溶剂和三不同的技术（浸提、索氏和回流）产生二十种不同的提取产物。通过高效液相色谱法检测不同的提取方法对钟花蒲桃的主要次生代谢产物的回收率。 结论 2 超临界流体萃取法优于传统溶剂提取法，省成本又省时。因此在工业使用上，超临界流体萃取法可替代传统溶剂提取法。 Syzygium campanulatum Supercritical fluid extraction Conventional solvent extractions Flavanones Chalcone Triterpenoids 钟花蒲桃 超临界流体萃取法 传统溶剂提取法 黃烷酮类化合物 查耳酮类化合物 三萜类化合物 Introduction 1 Syzygium campanulatum Korth is commonly found as an ornamental tree, primarily planted along roads and public places in Malaysia and Thailand. It is quite familiar as Kelat Paya in Malaysia (Nazarudin et al., 2010 2010 ). Traditionally, it is reported as a stomachic (Memon et al., 2014 2014 ). Scientifically, its n -hexane: methanol ( n -HEX:MeOH) extract is characterized as ( E )-2',4'-dihydroxy-6'-methoxy-3',5'-dimethylchalcone (DMC), ( 2S )-7-hydroxy-5-methoxy-6,8-dimethyl flavanone (HMDF), ( S )-5,7-dihydroxy-6,8-dimethyl-flavanone (HMF), betulinic acid (BA), and ursolic acid (UA) (Memon et al., 2014 2014 ; 2015 2015 ). The MeOH extract of S. campanulatum has been analyzed for anticolon cancer activity (Aisha et al., 2013 2013 ). In addition to S. campanulatum , flavanones, chalcones, and triterpenoids are also found as the most frequent secondary metabolites in other plants (Jäger et al., 2009 2009 ; Oskoueian et al., 2013 2013 ). Furthermore, these secondary metabolites possessed paramount pharmacological activities like anticancer (Wattenberg et al., 1994 1994 ), analgesic, anti-inflammatory (Kim et al., 2010 2010 ), antibacterial, antifungal, antiprotozoal (Gafner et al., 1996 1996 ), and antiviral (Havsteen, 2002 2002 ). Generally, non-polar solvents ( n -HEX and chloroform) and polar solvents (acetone (Ace) and ethanol (EtOH)) are reported for the extraction of aglycone flavonoids and flavonoids with glycones, respectively (Veličković et al., 2007 2007 ). Chalcones, being semi-polar compounds, are extracted with a mixture of polar and non-polar solvents (Memon et al., 2014 2014 ). Various methods of extraction, such as liquid solvent-based, ultrasound-assisted, pressurized liquid (Pinilla et al., 2014 2014 ), soxhletion (Bimakr et al., 2011 2011 ), heat reflux (Wong-Paz et al., 2014 2014 ), and supercritical fluid extractions (Lien et al., 2014 2014 ), are reported for maximum recoveries of flavonoids (He et al., 2005 2005 ) and triterpenoids (Domingues et al., 2012 2012 ) from various plants. In our present study, different conventional solvent extraction (CSE) and supercritical fluid extraction (SFE) techniques are performed and compared to achieve maximum recovery of abovementioned secondary metabolites from S. campanulatum leaves. However, to our knowledge, a comparative study between CSE and SFE methods for the recovery of major secondary metabolites from S. campanulatum leaves has not yet been thoroughly performed. The main purpose of this study is to propose a suitable quality control method for extracting these five major secondary metabolites (two flavanones, chalcone, and two triterpenoids) from S. campanulatum leaves for industrial scale applications. Furthermore, ethanol was found as a modifier because it is environmentally benign and relatively safe for human health (He et al., 2005 2005 ). Materials and methods 1 Plant materials 2 The fresh leaves of the S. campanulatum plant were collected in June 2015, from the main campus of the Universiti Sains Malaysia (USM), Penang, Malaysia. The plant was authenticated by the School of Biological Sciences, USM, where a voucher specimen was deposited (Ref. No. 11047). The green leaves of the S. campanulatum plant were washed under tap water, dried in an oven at 40 °C for 2‒3 d, and finally ground to fine powder using an electric grinder (Retsch, Germany). Chemicals 2 EtOH of food grade from chemical codex, MeOH, n -HEX, ethyl acetate (EA) of analytical grade, hydrochloric acid (HCl), sodium hydroxide (NaOH), acetonitrile (ACN), Ace, orthophosphoric acid, BA, and UA of high performance liquid chromatography (HPLC) grade were purchased from Sigma-Aldrich, Germany, while HMDF, HMF, and DMC were isolated from the S. campanulatum extract. The carbon dioxide (CO 2 ) of SFE grade contained in a high pressure dip tube cylinder was purchased from Kras Instrument & Services, Johor, Malaysia. Instruments 2 The lab-scale super critical (SC)-CO 2 unit consisted of an extraction vessel (100 ml), high-pressure pump (Supercritical 24, Lab Alliance, USA), modifier pump (Series II Pump, Lab Alliance, USA), automated back pressure regulator (Jasco, BP-2080 Plus), oven-U10 (Memmert) used as an extraction chamber, and a 99% pure CO 2 tank (Kras Instrument & Services, Johor, Malaysia). A pilot-scale SC-CO 2 extraction unit (SF5, Separex, France) with extraction vessel (5 L), Agilent HPLC 1260 system (USA), and ZORBAX Eclipse Plus Phenyl Hexyl (4.6 mm×250 mm, 5 μm) C-18 column (Agilent, USA) were also used. Methods of extraction 2 Lab-scale supercritical fluid extraction 3 For the pilot-scale SFE method, a preliminary method was first optimized at the lab scale. A lab-scale SC-CO 2 unit was designed for the extraction of five major secondary metabolites from dried powdered leaves of the S. campanulatum plant. Experimental conditions were optimized as: pressure (10, 20, and 30 MPa), temperature (40, 60, and 80 °C), and particle size (300, 500, and 700 μm) using EtOH as a modifier (5% of CO 2 ) for the first hour, followed by a second hour of dynamic extraction in a solvent with a flow rate of 4 ml/min and plant mass of 10 g. For comparing the effect of the modifiers, Ace and MeOH were used (5% of CO 2 ) having a pressure of 30 MPa, temperature of 40 °C, and particle size of 500 μm for the same experimental time as mentioned for EtOH. SFE crude extracts were collected in a vial (25 ml) and kept at 4 °C. Extracts were then evaporated to dryness using a rotary evaporator at 50 °C. The experiment was performed based on the design that was developed using Statistica® (Version 8.0, StatSoft Inc.) and Design Expert® (Version 6.0.4, Stat-Ease Inc.). The Box-Behnken design was applied to assess the effects of processing parameters on response variables for the optimization of the extraction. Pilot-scale supercritical fluid extraction 3 The pilot-scale extraction using SC-CO 2 (SF5, Separex, France) was performed according to the optimum results obtained from the Box-Behnken design. The pilot-scale SC-CO 2 was equipped with a high-pressure CO 2 pump, back pressure regulator, chiller, extraction vessel (5 L), and separators. The extraction system was controlled automatically. The S. campanulatum leaves were extracted using a modifier (EtOH) with dynamic extraction. Powdered dried leaves (1 kg) with 500-μm particle size of the S. campanulatum plant were placed and sealed tightly in an extractor vessel (5 L). The temperature of the vessel, pre-heater, and separator was set at 50 °C. Then, the vessel was inserted into its chamber. All manual valves were kept closed and CO 2 inlet was opened slowly until the pressure became stable. The desired pressure (30 MPa) and flow rate (35 g/min) were fixed. The extraction process was continued for the second hour of dynamic extraction by feeding the food-grade EtOH as a modifier at the ratio of 5% of CO 2 . Conventional solvent extraction 3 For CSE, the dried powdered leaves (200 g) were extracted using three conventional methods of extraction (maceration (MAC), reflux (REF), and soxhletion (SOX)) and ten different solvents of 2 L (water, 0.1 mol/L HCl, 0.1 mol/L NaOH (at 95‒ 100 °C), H 2 O:EtOH (1:1, v/v), H 2 O:MeOH (1:1, v/v), EtOH, MeOH, EA, n -HEX, and n -HEX:MeOH (1:1), at 65‒80 °C for 24 h). The crude extracts were then filtered and concentrated to dryness using a rotary evaporator at 50 °C. Analysis of secondary metabolites 2 The quantifications of HMDF, HMF, DMC, BA, and UA in crude extracts were performed using the HPLC method as previously described (Memon et al., 2015 2015 ). Briefly, analysis was performed using a C-18 reverse phase ZORBAX Eclipse Plus Phenyl Hexyl column (4.6 mm×250 mm, 5 μm). A mobile phase consisting of HPLC-grade ACN:Milli-Q H 2 O:H 3 PO 4 (60:40:0.01, v/v/v) was used. Isocratic elution was performed keeping a flow rate of 1 ml/min, at 30 °C, with a total run time 20 min. The injection volume was 10 μl and chromatograms were recorded at 210 nm. Calibration curves of HMDF, HMF, DMC, BA, and UA were plotted separately, using the already pure isolated compounds as standards. Experimental design and statistical analysis 2 In this study, the Box-Behnken design consisting of fifteen runs, three factors, and three levels was employed for constructing a polynomial model for optimization of the maximum yields of crude extract, each secondary metabolite (HMDF, HMF, DMC, BA, and UA), and total secondary metabolites (TSM) as dependent variables using Statistica®(Version 8.0) and Design Expert® (Version 6.0.4). The Box-Behnken design was selected as it generates fewer runs using three independent variables (Table 1 1 ). X 1 =pressure; X 2 =temperature; X 3 =particle size; Y =yield 2 Level 3 Independent variables 2 Dependent variable, Y (%) X 1 (MPa) X 2 (°C) X 3 (μm) –1 10 40 300 3 Maximize 0 20 60 500 1 30 80 700 3 Results and discussion 2 3.1 Conventional solvent extraction 2 For CSE, ten different solvents and three methods of extraction were optimized and the yield of the crude extracts was calculated as the mean of duplicated experiments (Table 2 2 ). All the crude extracts were quantified using the HPLC method for quantification of major secondary metabolites and the representative chromatograms were recorded (Figs. 1 1 and S1 S1 ). It was observed that a 0.1 mol/L HCl-REF method produced a higher yield of the crude extract (30%) than the n -HEX:MeOH (1:1)-SOX (25%). No. Extraction CE (%) HMDF (%) HMF (%) DMC (%) BA (%) UA (%) TSM (%) 1 H 2 O-REF 20.0 0 0 0 0 0 0 2 H 2 O-MAC 5.0 0 0 0 0 0 0 3 0.1 mol/L HCl-REF 30.0 0 0 0 0 0 0 4 0.1 mol/L NaOH-REF 26.0 0 0 0 0 0 0 5 EtOH:H 2 O (1:1)-SOX 12.0 0.05 0.02 0.05 0.8 0 0.92 6 EtOH:H 2 O (1:1)-MAC 7.5 0.05 0.03 0.06 0.8 0 0.94 7 EtOH:H 2 O (1:1)-REF 28.0 0.04 0.02 0.04 0.8 0 0.90 8 MeOH:H 2 O (1:1)-SOX 14.0 0.05 0.08 0.08 2.3 0 2.51 9 MeOH:H 2 O (1:1)-MAC 20.0 0.05 0.06 0.08 2.7 0 2.89 10 MeOH:H 2 O (1:1)-REF 27.0 0.05 0.09 0.10 2.4 0 2.64 11 EtOH-SOX 26.0 1.30 0.24 2.30 35.0 2.8 41.64 12 EtOH-MAC 14.0 1.00 0.17 2.80 40.0 3.5 47.47 13 EtOH-REF 20.0 1.00 0.18 2.60 40.0 3.1 46.88 14 MeOH-SOX 26.0 0.80 0.18 2.00 31.0 2.5 36.48 15 MeOH-MAC 26.0 0.80 0.18 2.40 39.0 2.5 44.88 16 MeOH-REF 21.0 0.70 0.17 2.10 33.0 2.3 38.27 17 n -HEX:MeOH (1:1)-SOX 25.0 3.00 0.52 8.97 34.0 2.8 49.29 18 n -HEX:MeOH (1:1)-MAC 26.0 1.20 0.19 4.50 19.0 3.8 28.69 19 n -HEX-MAC 1.2 1.10 0.50 15.80 7.5 11.0 35.90 20 EA-MAC 10.0 0.03 0.12 0.17 58.0 7.7 66.02 In the 0.1 mol/L HCl-REF crude extract, no HMDF, HMF, DMC, BA, or UA metabolite was recovered due to their insolubilities in water (Fig. 1a 1a ). The n -HEX:MeOH (1:1)-SOX extract produced maximum recoveries of HMDF, HMF, DMC, BA, and UA (3.00%, 0.52%, 8.97%, 34.0%, and 2.8%; Fig. 1b 1b ). No recovery of TSM was obtained using H 2 O, 0.1 mol/L HCl or NaOH solvents. With EtOH:H 2 O (1:1) and MeOH:H 2 O (1:1), there was no recovery of UA (Figs. 1c and 1d 1c and 1d ). The HMDF, HMF, DMC, and BA were recovered at 0.04%–0.05%, 0.02%–0.09%, 0.04%–1.00%, and 0.8%–2.7%, respectively. (a) 0.1 mol/L HCl-REF; (b) n -HEX:MeOH (1:1)-SOX; (c) EtOH:H 2 O (1:1)-SOX; (d) MeOH:H 2 O (1:1)-SOX; (e) n -HEX-MAC; (f) EA-MAC EtOH and MeOH recovered HMDF, HMF, DMC, BA, and UA at 0.7%–1.3%, 0.17%–0.24%, 2.00%–2.80%, 31.0%–40.0%, and 2.3%–3.5%, respectively (Fig. S1 S1 ). Fig. 1e 1e shows a higher yield of DMC (15.80%) from n -HEX-MAC. EA-MAC recovered the maxima of BA (58.0%), while HMDF, HMF, DMC, and UA recovered 0.03%, 0.12%, 0.17%, and 7.7%, respectively (Fig. 1f 1f ). In all twenty CSE techniques, the maxima of HMDF, HMF, DMC, BA, and UA at 1.20%–3.00%, 0.19%–0.52%, 4.50%–8.97%, 19.0%–34.0%, and 2.8%–3.8% were recovered using the n -HEX:MeOH (1:1) SOX and MAC techniques. The data of maximum recovery obtained by the CSE technique is summarized in Table 2 2 . 3.2 Supercritical fluid extraction 2 3.2.1 Preliminary study 3 For SFE, the correlation between parameters was observed at the central point pressure of 30 MPa, and with particle size of 500 μm. The temperature at 40 °C and modifiers Ace, MeOH, and EtOH were used to find a suitable extraction time and modifier. There was an increase in the extraction rate using EtOH as a modifier until 90 min, followed by a 30-min period of a falling extraction rate. After 110 min, no significant increase in the extraction of major secondary metabolites was observed. The 120 min experimental time was optimized with using EtOH as a modifier. 3.2.2 Optimization of SFE using the Box-Behnken experimental design 3 The Box-Behnken experimental design was developed to optimize the SFE method for maximum recovery of major secondary metabolites from the S. campanulatum leaves. The various parameters of design and recovery are summarized in Table 3 3 . 2 Run 3 Design 2 CE (%) 2 HMDF (%) 2 HMF (%) 2 DMC (%) 2 BA (%) 2 UA (%) 2 TSM (%) X 1 X 2 X 3 1 −1 −1 0 0.5 2.08 0.53 10.2 11.49 2.33 26.65 2 1 −1 0 1.1 3.31 0.87 18.0 24.79 4.96 51.98 3 −1 1 0 0.1 0.73 0.19 4.6 0 0 5.55 4 1 1 0 1.3 2.26 0.58 12.8 21.31 9.25 46.25 5 −1 0 −1 0.2 0.88 0.32 5.9 13.83 2.25 23.19 6 1 0 −1 0.8 2.37 0.64 12.9 21.84 3.23 41.00 7 −1 0 1 0.2 0.98 0.30 8.0 11.13 1.35 21.77 8 1 0 1 0.8 3.00 0.80 17.2 20.79 2.96 44.80 9 0 −1 −1 0.8 2.02 0.55 11.7 21.04 3.23 38.56 10 0 1 −1 1.7 1.88 0.54 8.2 8.16 3.34 22.12 11 0 −1 1 0.8 2.37 0.62 12.2 9.46 3.04 27.77 12 0 1 1 1.6 2.09 0.50 11.1 6.98 2.07 22.82 13 0 0 0 1.8 2.19 0.60 12.2 9.98 4.04 29.09 14 0 0 0 1.8 2.04 0.58 13.0 9.89 4.26 29.79 15 0 0 0 1.8 2.11 0.63 12.0 9.93 3.98 28.68 16 0 0 0 1.7 2.34 0.65 11.8 9.90 4.00 28.88 17 0 0 0 1.7 2.29 0.59 12.0 9.85 3.76 29.56 3.2.3 Statistical analysis 3 The analysis of variance (ANOVA) was performed to fit the model for each variable. The statistical analysis for the regression of coefficients of different factors in all models is shown in Table 4 4 . It was observed that pressure ( X 1 ) significantly influenced yields of crude extract, HMDF, HMF, DMC, BA, UA, and TSM in the linear model with positive coefficient values, whereas crude extract and BA also achieved similar results in their quadratic forms. This reflects the fact that the maximum yields of crude extract, HMDF, HMF, DMC, BA, UA, and TSM were achieved when pressure was set at its highest level, approximately 30 MPa (Fig. 2 2 ). P value of < 0.05 is considered significant. CE: crude extract 2 Effect on yield 7 Coefficients 7 P CE HMDF HMF DMC BA UA TSM CE HMDF HMF DMC BA UA TSM Intercept 1.760 2.190 0.610 12.210 9.91 4.01 29.20 0.005 0.005 0.004 0.000 0.002 0.076 0.001 X 1 0.370 0.780 0.190 4.040 6.53 1.81 13.36 0.006 0.000 0.000 0.000 0.000 0.005 0.000 X 2 0.190 −0.350 −0.095 −1.930 −3.79 0.14 −6.03 0.088 0.012 0.007 0.000 0.004 0.774 0.003 X 3 −7.50×10 −3 0.160 0.021 1.250 −2.06 −0.33 −0.96 0.940 0.170 0.436 0.008 0.057 0.499 0.500 X 1 2 −0.880 −0.190 −0.053 −0.300 4.99 –0.17 4.14 0.000 0.231 0.182 0.551 0.005 0.793 0.062 X 2 2 −0.140 0.092 −0.015 −0.480 −0.50 0.30 −0.73 0.337 0.548 0.685 0.352 0.701 0.651 0.707 X 3 2 −0.390 −0.200 −0.042 −0.890 2.00 −1.39 −0.65 0.022 0.220 0.270 0.104 0.153 0.065 0.738 X 1 X 2 0.150 0.075 0.012 0.097 2.00 1.65 3.84 0.308 0.631 0.741 0.848 0.162 0.038 0.085 X 1 X 3 −2.50×10 −3 0.130 0.045 0.560 0.41 0.16 1.31 0.985 0.404 0.560 0.293 0.757 0.816 0.518 X 2 X 3 –0.018 −0.035 −0.028 0.610 2.60 −0.27 2.87 0.901 0.821 0.475 0.257 0.082 0.691 0.178 15 R 2 0.916 0.913 0.916 0.963 0.934 0.798 0.948 − − − − − − − R 2 adj 0.808 0.801 0.809 0.917 0.851 0.538 0.882 − − − − − − − On the other hand, the yield of crude extract decreased with the increase in pressure ( X 1 ). Temperature ( X 2 ) significantly influenced the yields of HMDF, HMF, DMC, BA, and TSM in their linear form and UA in its quadratic form. It can be observed that the decrease in temperature increased the yields of HMDF, HMF, DMC, BA, and TSM (Figs. 2b, 2c 2b, 2c , 2d, 2e 2d, 2e , and 2g 2g ). The increased yields of DMC in its linear form and crude extract in its quadratic form were significantly influenced by particle size ( X 3 ). The coefficient of determination ( R 2 ) and the adjusted coefficient ( R 2 adj ) of all models are presented in Table 4 4 . 3.2.4 Pilot-scale supercritical fluid extraction 3 The pilot-scale SFE method at pressure 30 MPa and temperature 40 °C and without a modifier produced a lower yield (0.8%) of crude extract as compared with that using EtOH as a modifier (1.0%). In the first hour of extraction without a modifier, HMDF, HMF, DMC, and BA were recovered at 3.1%, 0.98%, 21.05%, and 0.40%, respectively, while UA was not recovered without a modifier. In the first hour of extraction without a modifier, HMF and DMC were recovered at their highest yields (Fig. 3a 3a ). During the first hour of extraction with a modifier, the highest yields of HMDF and UA were observed (Fig. 3b 3b ). In the second hour extraction, the maximum yield was observed for BA. Table 5 5 summarizes the yield for the pilot-scale SFE. All respective chromatograms are shown in Fig. S2 S2 . (a) The first hour extraction without a modifier at pressure 30 MPa, temperature 40 °C, and particle size 500 μm; (b) The first hour extraction with a modifier at pressure 30 MPa, temperature 40 °C, and particle size 500 μm No. Extraction CE (%) HMDF (%) HMF (%) DMC (%) BA (%) UA (%) TSM (%) 1 The first hour without modifier 0.8 3.1 0.98 21.05 0.40 0 25.53 2 The first hour with modifier 1.0 5.7 0.96 18.07 45.20 4.6 74.53 3 The second hour with modifier 1.0 4.9 0.89 17.22 57.35 4.5 84.86 4 Conclusions 1 S. campanulatum leave extracts produced a higher yield of TSM by different CSE methods. High recoveries of HMDF (3.00%) and HMF (0.52%) were obtained using n -HEX:MeOH-SOX, DMC (15.80%) and UA (11.0%) using n -HEX-MAC, and BA (58.0%) using EA-MAC. In the pilot-scale SFE, the highest recoveries of HMF and DMC were observed within the first hour of extraction without a modifier (EtOH). The maximum recoveries of HMDF and UA were obtained during the first hour of extraction with a modifier. In the second hour of SFE extraction with a modifier (EtOH), BA was recovered at its highest yield. Using the lab-scale and pilot-scale SFE methods allows us to postulate that EtOH used as a modifier has a significant effect on the recovery of secondary metabolites from dried leaves of S. campanulatum . From the present study, it can be concluded that the pilot-scale SFE method is better than CSE in terms of less consumption of plant raw material, solvent, and time. The CSE methods are not constantly acceptable for industrial applications due to more time required for extraction, consumption of hazardous and expensive solvents, and more use of raw material and labor. Therefore, the developed SFE method can be an alternative extraction method to CSE for extraction of secondary metabolites from the leaves of the S. campanulatum plant. Acknowledgements 2 The authors are thankful to the Universiti Sains Malaysia for providing financial support. 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