Journal of Applied Research on Medicinal and Aromatic Plants xxx (xxxx) xxx
Please cite this article as: Soon Hong Soh, Journal of Applied Research on Medicinal and Aromatic Plants, Available online 22 August 2020
2214-7861/© 2020 Elsevier GmbH. All rights reserved.
Optimized extraction of patchouli essential oil from Pogostemon cablin Benth. with supercritical carbon dioxide
Soon Hong Soh
a,b, Akshay Jain
a, Lai Yeng Lee
b,1, Sundaramurthy Jayaraman
a,*
aEWT Centre of Innovation, Ngee Ann Polytechnic, 535 Clementi Road, Singapore, 599489, Singapore
bNewcastle University in Singapore, 537 Clementi Road, Singapore, 599493, Singapore
A R T I C L E I N F O Keywords:
Supercritical fluid extraction Carbon dioxide
Patchouli Antioxidant activity Taguchi method
A B S T R A C T
In this work, supercritical fluid extraction (SFE) was employed to fully utilize patchouli plant including the stems to extract patchouli oil. In the conventional extraction by steam distillation, patchouli stems are often underu- tilized and discarded as waste. Using Taguchi’s orthogonal array method, the effect of pressure (10–20 MPa), temperature (35–45◦C) and CO2 flowrate (40–80 g/min) on SFE yield, antioxidant activity and patchoulol concentration from patchouli plant were evaluated. The SFE process conditions were optimized based on the selective yield of patchoulol as patchoulol is generally considered as an indicator of the patchouli oil quality. SFE with the optimized pressure of 15 MPa, temperature of 45◦C and CO2 flow rate of 60 g/min obtained yield, antioxidant activity and patchoulol concentration of 3.51 %, IC50:1.21 mg/mL and 0.2953 g patchoulol/g oil respectively. The results were compared with steam distillation where SFE shown significantly improved yield and antioxidant activity over the distillation process.
1. Introduction
Patchouli oil is a valuable essential oil obtained from the leaves, stems and flowers of patchouli (Pogostemon cablin Benth.), an herbal plant extensively cultivated in Brazil, China and Indonesia. Patchouli oil has great commercial importance in the international market and is extensively used in food, clinical and cosmetics applications (Manoj et al., 2012; Swamy and Sinniah, 2015). Furthermore, there is more demand for patchouli oil due to its unavailability of synthetic substitu- tion (Farooqi et al., 2003). The composition of patchouli oil is unique and complex due to its large number of different sesquiterpenes in contrast to a blend of different mono-, sesqui- and di-terpene compounds (Deguerry et al., 2006). Patchouli alcohol (C15H26), also known as patchoulol, is one of the main chemical components responsible for the strong, long-lasting fragrant aroma of the oil. Patchoulol possesses insecticidal, neuroprotective, anti-influenza, anti-inflammatory and anti-tumorigenic activities (Feng et al., 2019; Jeong et al., 2013). Other minor constituents such as caryophyllene, α, β-patchoulene, pogostol and seychellene are also present (van Beek and Joulain, 2018).
In recent years, the phytochemical constituents of plants have garnered much attention due to their potential application in the
nutraceutical and pharmaceutical industries. The use of plant extracts as natural antioxidants is a field of growing interest as synthetic antioxi- dants butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT) are now suspected to be potentially harmful to human health (Amorati et al., 2013). However, the plants possess antioxidant activity because they contain phytochemicals like phenols, thiols, carotenoids and tocopherols, which reduce oxidative damages to biomolecules by modulating reactive free radicals (Shui and Leong, 2006). As such, they are part of the daily food intake in many regions of the world as natural sources of flavouring and preservatives. Plants and herbs are, in general, harmless sources for obtaining natural antioxidants. Adding plant ex- tracts to edible products may thus replace synthetic antioxidant food additives as a possible alternative to prevent rancidity and prolong shelf life (Amorati et al., 2013). Patchouli oil is known to contain antioxidant properties that could be beneficial for nutraceutical application (Manoj et al., 2012; Wei and Shibamoto, 2007) and is used as a flavour ingre- dient in major food products including alcoholic and non-alcoholic beverages. Minimal concentration (2 mg/kg) of oil is sufficient to flavour foods, beverages, candy and baked products (Das, 2015).
Commercial patchouli oil is generally obtained by steam distillation of dried patchouli leaves (Kusuma and Mahfud, 2017; Yahya and Yunus,
* Corresponding author.
E-mail address: [email protected] (S. Jayaraman).
1 Current affiliation: National University of Singapore, 4 Engineering Dr 4, Singapore 117585.
Contents lists available at ScienceDirect
Journal of Applied Research on Medicinal and Aromatic Plants
journal homepage: www.elsevier.com/locate/jarmap
https://doi.org/10.1016/j.jarmap.2020.100272
Received 29 April 2020; Received in revised form 10 August 2020; Accepted 16 August 2020
2013). Patchouli stems contained much lower essential oil which is often characterized with different chemical composition (Henderson et al., 1970; van Beek and Joulain, 2018) and thus are often underutilized and discarded. Many extraction studies pertaining to patchouli oil dealt with only patchouli leaves, excluding the stems (Ambrose et al., 2013;
Donelian et al., 2009; Kusuma and Mahfud, 2017; Shah et al., 2017;
Yang et al., 2013). In the steam distillation of patchouli oil, shortcom- ings include the use of elevated temperature and long extraction time which leads to high energy consumption (Kusuma and Mahfud, 2017).
To circumvent these drawbacks, supercritical fluid extraction (SFE) using carbon dioxide offers an attractive alternative. The application of SFE for the recovery of essential oil and seed oil is gaining immense popularity over the years (de Melo et al., 2014; Fornari et al., 2012;
Reverchon and De Marco, 2006; Sahena et al., 2009). This is attributed to the relatively low critical temperature (31.1◦C) and pressure (7.39 MPa), inert properties, non-toxicity, non-flammability and low cost of supercritical CO2. Furthermore, SFE allows low temperature and solvent-free extraction which is especially beneficial in cosmetics, foods, and pharmaceutical products where there are more stringent re- quirements. Moreover, supercritical CO2 has a polarity comparable to liquid pentane which makes it compatible for the solubilization of lipophilic compounds such as essential oil as well as seed oil (Capuzzo et al., 2013).
Design of experiments (DOE) are often used in the optimization and investigation of effect of different operating parameters of an extraction process. DOE consist of numerical and statistical approach encompass- ing the fitting of empirical models that analyze the influence of variables on a response, leading to their ranking and to discard the nonsignificant variables (Bezerra et al., 2008). SFE studies have been carried out using DOE such as the Taguchi method (Ansari and Goodarznia, 2012; Guan et al., 2007; Salea et al., 2017; Subroto et al., 2017) and the response surface methodology-central composite design (RSM-CCD) (Ara and Raofie, 2016; Sodeifian et al., 2016). RSM-CCD is a compilation of sta- tistical and mathematical approaches for modelling, problem analysis development, modification and optimization of various processes whereas the Taguchi method is a unique statistical approach that opti- mize the processes by searching the suitable operating process condi- tions. RSM requires a larger number of experiments while Taguchi uses the least number of experiments to determine the optimum process condition (Tan et al., 2017).
Limited studies can be found on the SFE of patchouli oil and most of them have been carried out on patchouli leaves (Donelian et al., 2009;
Soh et al., 2019; Xiong et al., 2019). SFE of essential oil from patchouli stems have been reported by Liu et al. (Liu et al., 2008). However, their work mainly focuses on the yield of patchouli oil. In this study, the optimal conditions of SFE for the selective extraction of patchoulol from patchouli plant are identified. The influence of operating parameters (pressure, temperature, CO2 flow rate) of SFE on the yield, antioxidant activity and patchoulol concentration will also be described using the Taguchi method. The aim of this research is to evaluate the feasibility of recovering essential oil from the whole patchouli plant, fully utilizing the stems to minimize waste, by comparing experimental results to conventional steam distillation to assess the competitiveness of patch- ouli oil obtained by SFE.
2. Materials and methods 2.1. Materials and chemicals
Dried patchouli plant, comprising of 50 % leaf to stem ratio, was obtained from Indonesia. The moisture content of the plant was 10.60±0.07 %, measured using a halogen heating moisture analyser (MOC63 u UniBloc, Shimadzu). The leaves and stems were separated manually and ground separately in an electric blender. The ground materials were passed through a 0.6 mm stainless steel sieve. Samples were packed and stored in a dry cabinet at room temperature until
utilization. SFE optimization experiments were carried out with a mixture comprising of 1:1 ratio of ground leaves and stems. Liquid CO2 with a purity of 99.5 % was obtained from Air Liquide Singapore Pte Ltd.
Hexane and methanol of analytical grades were obtained from Fischer Scientific Pte Ltd (Singapore). Patchouli alcohol of ≥95 % purity was obtained from Cayman Chemical. Anhydrous sodium carbonate, sodium sulphate and 2,2-diphenyl-1-picrylhydrazyl (DPPH) were procured from Sigma-Aldrich Pte Ltd (Singapore).
2.2. Supercritical fluid extraction process
SFE experiments were carried out using a customized supercritical fluid extractor with CO2 recycle system (Model SFE 1000 System, Waters Corporation, USA). Fig. 1 shows a schematic diagram of the SFE setup (Soh et al., 2019). 1 L extraction vessel was loaded with 200 g ground material and packed with glass beads. Liquid CO2 was preheated and delivered to the extraction vessel using a high-pressure liquid pump.
Extraction pressure and temperature varied from 10 to 20 MPa and 35–45◦C, respectively. The operating pressure of the extraction vessel was controlled by an automated backpressure regulator (ABPR). The pressure of the collection vessel was maintained at 6.6 MPa by a manual backpressure regulator (MBPR) and temperature was maintained at 40◦C, for the recycling of CO2. At this stage, CO2 reverts to its gaseous state and the plant extracts precipitate to the bottom of the vessel.
Temperature in the extraction and collection vessels were maintained using band heaters (±1◦C). A 30 min static extraction period was fol- lowed by 60–120 min of dynamic extraction, depending on the CO2 flow rate varying from 40 to 80 g/min, to give a final solvent-to-feed ratio of 24. At the end of each experiment, the plant extract was collected from the collection vessel using an Erlenmeyer flask. After collection, the system was purged with CO2 to obtain residues. The plant extracts were dehydrated with anhydrous sodium sulphate, weighed and stored in a refrigerator for further analysis. The yield was gravimetrically calcu- lated by dividing the weight of plant extract by the amount of solid raw material.
2.3. Steam distillation process and equipment
100 g of dried patchouli plant (1:1 ratio of ground leaves and stems) was placed in a biomass flask, connected between a filled boiling flask and condenser. Water in the boiling flask was heated and the generated water vapour penetrated through the plant material, carrying patchouli oil to the condenser. The oil-water vapour was condensed using a recirculating chiller and the oil was separated from water using a separator funnel. The steam distillation process was carried out for 5 h for a complete extraction. Residual water was removed from patchouli oil by drying with anhydrous sodium sulphate. The dehydrated essential
Fig. 1.Supercritical fluid extraction setup (SFE 1000 system, Waters) C1: Compressed CO2 cylinder; E1: Condenser; E2: Electric preheater; P1: High pressure liquid pump; P2: Automated backpressure regulator; P3: Manual backpressure regulator V1: CO2 recycler; V2: Extraction vessel; V3: Collec- tion vessel.
oil was weighed and stored in a refrigerator for further analysis.
2.4. DPPH radical scavenging activity
Different dilutions of extracts (2 ml) were added to 2 ml of 2, 2- diphenyl-1-picrylhydrazyl (4 mg/100 ml methanol). Absorbance was measured at 517 nm after 30 min. Radical–scavenging ability was calculated as IC50 using the following equation:
I%=Ab− As
Ab
×100 (1)
Where Ab and As are the absorbance values of the DPPH blank solution and tested samples, respectively. IC50 (mg/mL) is the effective concen- tration of plant extract at which DPPH radicals are scavenged by 50 % and hence lower IC50 values mean better antioxidant activity. This value was determined by interpolation and linear regression.
2.5. GC–MS and GC-FID analysis
GC–MS analysis was performed using coupled gas chromatograph with mass spectroscopy (Clarus 600 GC/MS) equipped with a HP-5MS column (30 m x0.25 mm i.d., film thickness 0.25μm, Agilent). The oven temperature was held constant at 60◦C for 1 min and then the temperature was increased to 220◦C with the temperature increase rate of 30◦C/min. The final oven temperature was then kept constant for 4 min. The injector, transfer line and detector temperatures were 250, 240 and 230◦C, respectively. Ionization energy was 70 eV and the flow rate of carrier gas (helium) was 1 ml/min. The samples (1% v/v in hexane) was injected into the GC by split mode with a split ratio of 1/
100. Identification of patchoulol was based on comparison of the mass spectra obtained in the gas chromatograph with that obtained from the GC–MS library and from literature (van Beek and Joulain, 2018).
GC-FID analyses were performed using a gas chromatograph (Shi- madzu GC-2010) equipped with a flame ionization detector (FID) and a Rtx-624 column (60 m x0.32 mm i.d., film thickness 1.8μm, Restek).
Oven temperature was 60◦C for 1 min, before programmed heating to 220◦C at a rate of 30◦C/min and held at 220◦C for 24 min. Injector and detector temperatures were 220◦C. The carrier gas, helium, was adjusted to a linear velocity of 24 ml/min. The samples (1% v/v in hexane) were injected into the GC by split mode with a split ratio of 5.
Quantification of patchoulol was carried out by evaluating the correla- tion between the amount of patchouli alcohol and the peak area after analysing a series of standard solutions of patchouli alcohol in hexane (0.2–3 parts per million).
2.6. Design of experiments and statistical analysis
Taguchi experimental design was applied to determine the influence of SFE factors on yield, patchoulol and antioxidant activity. Three fac- tors of SFE include pressure (P: 10–20 MPa), temperature (T: 35–45◦C)
and CO2 flow rate (Q: 40–80 g/min). The effect of SFE parameters on yield, antioxidant activity and patchoulol content were evaluated. The statistical analysis of the data obtained from lab experiments were performed with analysis of variance (ANOVA), which was used to study the significance of control factors on yield and antioxidant activity of patchouli oil. P-value of less than 0.05 was determined as significant.
3. Results and discussion
Table 1 summarizes the experimental conditions and results ob- tained for each SFE run. Experimental results showed that yield ranged from 3.20 to 4.00 % yield, antioxidant activity ranged from 0.42 to 1.92 mg/ml IC50 values, patchoulol concentration of 0.161 – 0.281 g patchoulol/g oil and an extracted total patchoulol amount of 1.338–1.885 g.
Analysis of variance (ANOVA) with 95 % confidence interval was conducted to statistically analyse the influence of pressure, temperature and CO2 flow rate on the yield, antioxidant activity and patchoulol content as shown in Table 2. Minitab v.18 statistical software was used for the statistical analysis. In terms of significant effect, pressure had the greatest effect on yield and antioxidant activity whereas for patchoulol concentration, temperature is the most influencing factor. ANOVA with 95 % confidence interval indicated that different levels of pressure had a significant effect (P<0.05) whereas different levels of temperature and CO2 flow rate showed insignificant effects (P>0.05) on yield. It also shows that different levels of pressure and temperature had a significant effect on antioxidant activity while different levels of CO2 had shown
Table 1
Three-level orthogonal array design and experimental results for the SFE from patchouli plant (mean±standard deviation values (SD); SD=
̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅
∑ (X− X)2 n− 1
√√
√√
; X = individual value; X =sample mean; n =sample size=3).
Run P (MPa) T (◦C) Q (g/min) Yield (%) IC50 (mg/mL) Patchoulol conc. (g/g oil) Patchoulol total (g)
1 10 35 40 3.50±0.25 0.98±0.16 0.1931±0.0069 1.338±0.048
2 10 40 60 3.41±0.19 1.54±0.17 0.2512±0.0057 1.696±0.039
3 10 45 80 3.20±0.24 1.92±0.13 0.2809±0.0051 1.780±0.033
4 15 35 60 3.80±0.26 0.46±0.02 0.2502±0.0081 1.883±0.061
5 15 40 80 3.55±0.22 0.68±0.03 0.2629±0.006 1.848±0.043
6 15 45 40 3.51±0.39 1.25±0.13 0.2712±0.0057 1.885±0.040
7 20 35 80 4.00±0.33 0.42±0.03 0.1609±0.0039 1.274±0.031
8 20 40 40 3.95±0.25 0.58±0.04 0.2123±0.0045 1.660±0.035
9 20 45 60 3.60±0.46 1.03±0.12 0.2544±0.0063 1.814±0.045
Table 2
Analysis of variance (ANOVA) table for the SFE of patchouli oil (yield, antiox- idant activity and patchoulol content).
Source DF Adj SS Adj MS F-Value P-Value
ANOVA for yield
Pressure 2 0.345739 0.172869 19.38 0.049
Temperature 2 0.164272 0.082136 9.21 0.098
CO2 flowrate 2 0.007906 0.003953 0.44 0.693
Residual Error 2 0.017839 0.008919
Total 8 0.535756
ANOVA for antioxidant activity
Pressure 2 1.11995 0.559973 24.82 0.039
Temperature 2 0.92890 0.464449 20.58 0.046
CO2 flowrate 2 0.00993 0.004965 0.22 0.820
Residual Error 2 0.04513 0.022565
Total 8 2.10390
ANOVA for patchoulol content
Pressure 2 0.004169 0.002084 6.89 0.127
Temperature 2 0.006917 0.003458 11.43 0.080
CO2 flowrate 2 0.001077 0.000538 1.78 0.360
Residual Error 2 0.000605 0.000302
Total 8 0.012767
DF: Degree of freedom; Adj SS: Adjusted sums of squares; Adj MS: Adjusted means of squares.
insignificant effect. In terms of patchoulol concentration, the different levels of all three factors (pressure, temperature and CO2 flow rate) had shown no significant effect.
3.1. Effect of pressure, temperature and CO2 flow rate on yield and antioxidant activity of patchouli oil
The effects of pressure (10–20 MPa), temperature (35–45◦C) and CO2 flow rate (40–80 g/min) on yield and antioxidant activity is high- lighted in Fig. 2. Fig. 2A shows that the yield increased significantly with increase of pressure which is consistent in other SFE works (Salea et al., 2017; Sodeifian and Sajadian, 2017). This was attributed to the enhanced density of CO2 at high pressure, which promoted the solvation power of the supercritical fluid. The increase in pressure also accelerated solute and solvent mass transfer in supercritical extractor vessel system and increased the solubility of oil (Reverchon and De Marco, 2006).
Similarly, a positive effect on antioxidant activity is shown with increasing pressure. As CO2 densities increased, the distance between the molecules of solutes and solvent decreased. Therefore, more inter- action occurred between the solutes and CO2 which permitted higher specific extraction of polar and bioactive compounds (Sodeifian and Sajadian, 2017). At higher pressures, the enriched extraction of bioac- tive compounds, such as phenols and flavonoids, contributed to the in- crease in antioxidant activity (Machado et al., 2015).
On the contrary, the effect of temperature on yield was detrimental (Fig. 2B). At isobaric conditions, an increase in temperature reduced the
density of supercritical CO2, hence reducing the solvent power of the fluid. However, higher temperature increased vapour pressure of the solutes making the oil easier to extract (Espinosa-Pardo et al., 2014).
These two competing factors is dependent on extraction conditions, where above the crossover pressure, the effect of temperature on density became less than that of solute vapour pressure. Therefore, the yield tends to increase with temperature. Below the crossover pressure, the effect of temperature on density outweighed that of solute vapour pressure (Macías-S´anchez et al., 2005). Previous studies have shown that crossover phenomenon was observed at high pressure (≥20 MPa), while extraction temperature was increased (Espinosa-Pardo et al., 2014). In this study, since pressure was set at relatively low pressure (10–20 MPa), the crossover phenomenon was not observed. Hence, the effect on temperature on density outweighed that of solute vapour pressure. In addition to the reduced density of the supercritical fluid solvent, the increased temperature caused heat sensitive antioxidants and bioactive compounds to degrade (R´eblov´a, 2012). This led to an adverse effect on antioxidant activity.
Fig. 2C shows no apparent effect of CO2 flow rate on yield and antioxidant activity. At low solvent flow rates, the mass transfer resis- tance limited the amount of solute transported into the bulk of the sol- vent and the supercritical CO2 exited the extractor unsaturated. As the flow rate was increased, a greater amount of CO2 passed through the extractor, decreasing the mass transfer resistance until the exiting sol- vent was saturated. However, excessive CO2 flow rate reduced the residence time, causing the system to deviate from equilibrium, and the solvent exited the extractor unsaturated despite the increased mass transfer rate (Kumoro and Hasan, 2008). In this study, a solvent to feed ratio of 24 was used and solvent saturation was attained. Therefore, the effect of CO2 flow rate displays no significant change on yield. An identical trend was observed in the SFE of candlenut oil using a solvent to feed ratio of 48 (Subroto et al., 2017). Meanwhile, Fig. 2C shows no significant change in antioxidant activity when CO2 flow rate was var- ied. A change in CO2 flow rate did not alter the physical properties of CO2, namely density and hence solubility. As a result, there was no effect of flow rate on the antioxidant activity of the plant extract. A similar finding was reported in the study of SFE of lycopene from tomato skins by Yi et al. (Yi et al., 2009).
3.2. Effect of pressure, temperature and CO2 flow rate on patchoulol concentration
The effect of pressure, temperature and CO2 flowrate on the SFE of patchoulol is highlighted in Fig. 3. Fig. 3A shows that an increase of pressure from 10 to 15 MPa enhanced patchoulol extraction. Donelian et al. (Donelian et al., 2009) reported a similar trend where an increase in pressure from 8.5 MPa to 14 MPa led to a greater patchoulol con- centration. However, in this study a further increase in pressure from 15 MPa to 20 MPa led to a decrease in patchoulol extraction. The increased solvent strength of supercritical CO2 decreased the extraction selectivity, promoting undesired co-extraction of non-volatile com- pounds, such as fatty acids and other lipids (Chen et al., 2018). As a result, the concentration of volatile patchoulol is reduced. Hamburger et al. (Hamburger et al., 2004) studied the effect of pressure on yield of extracted substances from three medicinal plants (marigold, hawthorn and chamomile). They reported an increase in yield of non-volatile lipophilic compounds, such as faradiol esters, are achievable at pres- sures above 30 MPa. However, the extended pressure range appears not to increase further the yield of volatile constituents.
Fig. 3B shows an increase in patchoulol concentration with increasing temperature. For a non-volatile solute, elevated temperatures would result in lower extraction recovery due to a decrease in solubility.
Contrarily, for a volatile solute, there is a competition between its sol- ubility in CO2 and its volatility (Pourmortazavi and Hajimirsadeghi, 2007). In the case of patchoulol, the effect of temperature on its vapour pressure predominated that of supercritical CO2 density since it is a Fig. 2.Effect of A) pressure; B) temperature; C) CO2 flowrate on yield and
antioxidant activity of patchouli oil.
volatile solute. Furthermore, the reduced solvation strength of CO2 at increasing temperatures avoided undesired co-extraction of non-volatile compounds. Thus, an increase in patchoulol concentration was observed when temperature is raised from 35 to 45◦C. A similar trend was re- ported in the SFE of mushroom alcohol, where mushroom alcohol con- tent increased from 35 to 55◦C at isobaric conditions of 8.5 MPa (Chen et al., 2018).
An increase of patchoulol content was observed in Fig. 3C when CO2
flowrate was raised from 40 to 60 g/min, followed by a slight decrease when CO2 flowrate was further raised to 80 g/min. At CO2 flowrate of 40 g/min, the mass transfer resistance limited the amount of patchoulol transported into the bulk of the solvent whereas at CO2 flowrate of 80 g/
min led to a reduced residence time of CO2 in the extraction vessel. Both instances caused the CO2 to leave the extraction vessel unsaturated, leading to a decrease in patchoulol content. Therefore, a CO2 flow rate of 60 g/min would be optimal for the selective extraction of patchoulol.
3.3. Optimized SFE conditions and comparison with steam distillation The amount of patchoulol is generally considered as an indicator of the patchouli oil quality (Donelian et al., 2009). The application of higher pressures and lower temperatures, albeit advantages for the yield and antioxidant activity of patchouli oil extraction, led to significant co-extraction of non-volatile compounds and waxes (Gaspar, 2002) and, consequently, to extracts with lower patchoulol content. Hence, opti- mized pressure, temperature and CO2 flowrate based on patchoulol response were 15 MPa, 45◦C and 60 g/min respectively where at these
conditions, the lighter and more volatile patchoulol was selectively extracted. These data were subjected to Minitab v.18 statistical software package to predict the yield, antioxidant activity and patchoulol con- centration at proposed optimum condition. This methodology has been reflected in few earlier studies (Salea et al., 2014, Salea et al., 2017).
Verification experiment was also performed to check the accuracy of proposed optimum conditions. Table 3 shows that confirmation exper- iments have similar value to the predicted results and also compares the results between SFE and steam distillation.
In comparison with steam distillation (0.71 %), SFE from patchouli plant produced up to a five-fold yield increase (3.43 %). SFE is able to extract a wide range of volatile and non-volatile compounds, depending on the CO2 density whereas steam distillation hardly recovers non- volatile compounds. For instance, sclareol, an important component of clary sage, was usually recovered in only very small quantity in steam distillation due to its very high boiling point (Caissard et al., 2012).
Therefore, SFE obtained a significantly larger yield over steam distillation.
Table 3 also shows that patchouli oil produced by SFE gave higher antioxidant activity than steam distillation. Steam distilled patchouli oil contains mainly volatile compounds, which have generally low antiox- idant activity and can be limitedly applied in food industry as authentic antioxidants (Inanç and Maskan, 2013). During steam distillation of patchouli oil, the antioxidant activity was partially lost. The character- istics of target antioxidant compounds were affected by the extraction method as they depend on heat, light, oxygen, along with several other factors. The prolonged exposure of the patchouli oil to heat and light in steam distillation had degraded these active compounds and caused a major deterioration to the antioxidant activity (Inanç and Maskan, 2013;
Sodeifian and Sajadian, 2017). Meanwhile, SFE was performed in a closed extraction vessel in the absence of light and oxygen which minimized the degradation reactions of active compounds. Moreover, SFE was performed at mild temperatures in contrast to steam distillation and therefore degradation of heat sensitive compounds was minimal.
However, Table 3 shows that patchoulol concentration found in SFE is lower than that of steam distillation. As steam distilled oil contains mainly volatile compounds, the concentration of patchoulol is expected to be higher than that produced by SFE. According to Bureau of Indian Standards, good quality patchouli oil should possess 27.0–35.0 % patchouli alcohol (IS3398, 2003). Hence, the patchoulol concentration (~29.5 %) produced by SFE at the proposed optimized conditions of 15 MPa, 45◦C and 60 g/min was more than satisfactory. Nevertheless, the total amount of patchoulol recovered by SFE (2.073 g/200 g raw material) was significantly higher than steam distillation (0.316 g/100 g raw material).
4. Conclusion
The present study successfully applied Taguchi method to evaluate the effects of pressure, temperature and CO2 flowrate on yield, antiox- idant activity and patchoulol concentration in the SFE of essential oil from patchouli plant. An increase in pressure had a positive effect on yield and antioxidant activity of patchouli oil, whereas an increase in temperature had an adverse effect. In terms of patchoulol concentration, pressure had a positive effect when pressure is increased from 10 to 15 MPa, but a further increase in pressure to 20 MPa caused a negative effect. The increase in temperature had a positive effect on patchoulol concentration. CO2 flowrate had negligible effect on yield and antioxi- dant activity. However, an increase in patchoulol concentration was observed when CO2 flow rate was increased from 40 g/min to 60 g/min but a further increase in CO2 flow rate had shown a decrease in patch- oulol concentration. The optimized pressure, temperature and CO2 flow rate for maximizing patchoulol extraction within the experimental domain were 15 MPa, 45◦C and 60 g/min, respectively. The extent of the impact of variables on selective patchoulol extraction are as follows:
temperature>pressure>CO2 flowrate.
Fig. 3. Effect of A) pressure; B) temperature; C) CO2 flowrate on patchoulol (primary axis represents patchoulol concentration; secondary axis represents total patchoulol amount).
In conventional steam distillation processes for the extraction of patchouli oil, only leaves are used and the stems of patchouli plant are generally discarded as waste. Therefore, in this aspect SFE offers a better extraction alternative as it is able to fully utilize the whole patchouli plant, reflected by the superior yield and antioxidant activity obtained.
Furthermore, the significantly improved yield and antioxidant activity over steam distillation suggested that SFE of patchouli oil was more suitable for a range of food and pharmaceutical applications.
Declaration of Competing Interest The authors declare no conflict of interest.
Acknowledgements
The funding support by Ministry of Education, Singapore under the Translational Innovation Fund (MOE2015-TIF-2-G-051).
Appendix A. Supplementary data
Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.jarmap.2020.100272.
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Table 3
Yield, antioxidant activity and patchoulol content of patchouli oil obtained by SFE and steam distillation (mean±standard deviation values).
Experiment Yield (%) IC50 (mg/mL) Patchoulol conc. (g/g oil) Patchoulol total (g)
SFEa Predicted 3.43 1.24 0.3073 2.108
Actual 3.51±0.24 1.21±0.17 0.2953±0.0072 2.073±0.051
Steam distillation 0.71±0.05 >4 0.4464±0.0105 0.316±0.007
(For SFE, patchoulol total=g/200 g raw material while for steam distillation, patchoulol total=g/100 g raw material).
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