Optimization of supercritical fluid extraction and pressurized liquid extraction of active principles from Magnolia officinalis using the Taguchi design

Separation and Purification Technology 71 (2010) 293–301

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Optimization of supercritical fluid extraction and pressurized liquid extraction of active principles from Magnolia officinalis using the Taguchi design Emily L.C. Cheah, Paul W.S. Heng, Lai Wah Chan ∗ Department of Pharmacy, Faculty of Science, National University of Singapore, 18 Science Drive 4, Singapore 117543, Singapore

a r t i c l e

i n f o

Article history: Received 30 April 2009 Received in revised form 4 December 2009 Accepted 12 December 2009 Keywords: Biphenol Magnolia officinalis PLE SFE Soxhlet extraction

a b s t r a c t The effects of major variables of supercritical fluid extraction (SFE) and pressurized liquid extraction (PLE) processes on the recoveries of target compounds and activity of Magnolia officinalis (Magnolia) bark extracts were studied by employing the L9 (3)4 Taguchi design matrix. In SFE, the percentage of extractable solids (ES) and those of the active principles, honokiol and magnolol, were strongly affected by extraction pressure and temperature. Selectivity for magnolol over honokiol, as expressed by the magnolol:honokiol ratio, was found to be predominantly influenced by SFE pressure (>50%), with lowest pressure setting at 20 MPa resulting in approximately 35:1 selectivity in favour of magnolol. In comparison, the magnolol:honokiol ratio obtained by the exhaustive Soxhlet extraction was 10:1. Particle size was an insignificant factor in SFE but was a strong influence on PLE performance due to better penetrative ability of supercritical CO2 into the plant matrix. Although SFE extracts had lower quantitative recoveries compared to PLE and SFE, extracts obtained demonstrated greater antioxidant activity. © 2009 Elsevier B.V. All rights reserved.

1. Introduction There has been a resurgence of interest in botanicals as they are rich resources of new drugs or potentially useful compounds. Of these compounds, plant phenolics are the most attractive due to their antioxidant properties which are potentially useful in chemotherapy and anti-aging. To date, the extraction of the plant phenolics has been largely dominated by conventional solvent extraction techniques, such as percolation and Soxhlet extraction (SE). These techniques, however, possess several limitations that include prolonged extraction time and high solvent usage. In addition, extracts obtained by these techniques usually contain significant amounts of other substances besides the active principles and secondary purification of the extracts is necessary. New technologies based on compressed fluids, such as supercritical fluid extraction (SFE) and pressurized liquid extraction (PLE), have evolved from the need to circumvent the above limitations and to reduce the use of organic solvents. PLE has found increasing use in the extraction of bioactive principles from medicinal plants [1–5]. The extraction process employs a solvent at elevated temperature and pressure to increase extraction efficiency [6]. The elevated pressure improves solvent contact with the extraction bed as well as allow a larger range of temperatures to be employed in the extraction process, not limited by the

∗ Corresponding author. Tel.: +65 6516 3506; fax: +65 6775 2265. E-mail address: [email protected] (L.W. Chan). 1383-5866/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.seppur.2009.12.009

atmospheric boiling point of the solvent alone–an advantage over conventional solvent extraction processes. Increased temperature improves analyte solubility, reduces solvent viscosity and surface tension, disrupts matrix–analyte interaction and accelerates extraction kinetics [7–9], thereby improving extraction efficiency. In SFE, carbon dioxide (CO2 ) is commonly employed above its critical pressure and temperature, thus imparting the unique properties of gas-like diffusivity and liquid-like viscosity to the extracting fluid, enhancing its penetration into solid matrices [10]. When used neat (i.e. without the addition of an organic cosolvent), supercritical CO2 depressurizes at ambient conditions, leaving the extract free from undesirable solvent residues. SFE is also ideal for extraction of sensitive bioactive compounds as the enclosed system ensures the absence of both light and air, and CO2 has little or no reactivity with most compounds [9,11–15]. SFE possesses the unique ability to allow modulation of the supercritical CO2 solvent strength for selective isolation of particular components from the plant matrix. The chemometric approach applies a statistical method to study the effects of multiple variables simultaneously, saving cost, time, resource and labour. The Taguchi (orthogonal array) design is a type of fractional factorial design whereby the effects of variables at different levels and in different combinations are determined. The results obtained are interpreted by analysis of variance (ANOVA) or by direct comparison of the responses to the variables at different levels. The theory and methodology of orthogonal array designs for optimization of analytical procedures have been well explained by several researchers [16–21].

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Fig. 1. Chemical structures of (a) honokiol and (b) magnolol.

The bark of Magnolia officinalis is used in traditional Chinese medicine for the treatment of acute pain, cough and stomach ailments. The major active constituents of this bark are honokiol and magnolol (Fig. 1) which are biphenol isomers. These compounds are reported to confer potent pharmacological effects that include antioxidant [22–26], anti-inflammatory [27–30] and neuroprotective properties [26,31–33], protection against reperfusion injury [23,25,34–37], anti-cancer activity [38–40] as well as bacteriostatic and fungistatic effects [30,41–43]. The aim of the present work was to investigate the effects of major process parameters of SFE and PLE on the extraction of bioactives from Magnolia bark using the Taguchi approach. The performance of these extraction technologies was compared to that of the gold standard, SE. The goal was to obtain high selective recovery of the active principles while minimizing co-extraction of unwanted compounds. 2. Experimental 2.1. Herbal material Sliced and stripped Magnolia bark originating from Zhejiang, China (year of harvest 2004) was purchased from a local wholesaler (WHL Ginseng and Herbs Pte. Ltd., Singapore). The Magnolia bark was confirmed to be Magnolia biloba (Rehd. et Wils.) Cheng (or “houpo”) by Prof. Baolin Guo, Director of Herbarium of Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences, China. The bark was air-dried and milled to produce different batches with median mass diameter (MMD) of 683, 1003 and 1413 ␮m respectively (Table 1). 2.2. Chemical standards Honokiol, or 3 -(2-propenyl)-5-propyl-(1,1 -biphenyl)-2,4 -diol (ca. 99.9%, lot 050203) and magnolol, or 5,5 -di-2-propenyl-1,1 biphenyl-2,2 -diol (ca. 99.9%, lot 040720) were purchased from Seachem Co., Beijing, China. The standards were stored at 4 ◦ C until use. 2.3. Solvents, reagents and other materials Purified liquid carbon dioxide (99.8% purity, Soxal, Singapore) was used for SFE. n-Hexane (analytical grade, BDH, Poole, England) Table 1 Particle size distribution of comminuted Magnolia officinalis bark for extraction processes. D10 , MMD and D90 is the 10th, 50th and 90th percentile of the cumulative % undersize distribution plot. SX50 is the span value of the distribution curve and is calculated as (D90 − D10 )/MMD.

Size 1 Size 2 Size 3 a

MMD (␮m)

D10 (␮m)

D90 (␮m)

SX50 (␮m)

683.3 (37.9)a 1003 (37.9) 1413 (11.5)

120.0 (0) 156.7 (5.8) 193.3 (5.8)

1553.3 (35.1) 1936.7 (11.5) 2716 (28.9)

2.1 (0.1) 1.8 (0.1) 1.8 (0)

Numbers in parenthesis indicate standard deviation (n = 3).

was used for PLE and SE. Nitrogen gas (99.99% purity, Soxal, Singapore) was used for purging of the extraction cartridge in PLE. Methanol, acetonitrile (HPLC grade, Tedia Company, Fairfield, OH, USA) and o-phosphoric acid (Sino Chemicals, Singapore) were used for preparing the HPLC mobile phase. For reconstitution and dilution purposes in the antioxidant assay, methanol (analytical grade, Tedia Company, Fairfield, OH, USA) was used. The reagent for the antioxidant assay, 2,2-diphenyl-1-picryl-hydrazyl (DPPH) was purchased from Sigma–Aldrich, St. Louis, MO, USA. Glass wool was purchased from Poly Glass Fibre (Perai, Malaysia) and cellulose filters from Whatman (Kent, England). 2.4. Supercritical fluid extraction Supercritical fluid extractions were conducted using the SFT150 extraction system (Supercritical Fluid Technologies, Newark, DE, USA). Ten g of milled bark was packed into an extraction cartridge with an internal volume of 100 mL. Both ends of the extraction cartridge were packed with glass wool to improve dispersion of the supercritical CO2 as well as to reduce free void volume in the cartridge. Density of the pure CO2 was estimated using Bender’s equation of state based on the parameters listed by Brunner [44] while the Hildebrand solubility parameter was estimated using the modified equation by Giddings [45]. The estimates do not taken into account changes associated with dissolution of the extract in the supercritical CO2 . All extractions involved an initial static extraction phase of 60 min followed by dynamic extraction at a flow rate of 2 mL/min of liquid carbon dioxide. A variable restrictor valve, factory pre-set at 80 ◦ C, was used to control flow rate measured by a gas flowmeter (Dwyer, Michigan City, IN, USA) at the exhaust end of the collection vessel. The extracts obtained were collected in an empty collection vessel sealed with rubber septum and placed in an ice bath. After each experiment, the tubing and valves were flushed with methanol and the washings collected and added to the extracts. All extractions were carried out in triplicate. 2.5. Pressurised liquid extraction PLE was performed using a Dionex ASE 100 instrument (Dionex Corp., Sunnyvale, CA, USA). The system consisted of a solvent module, pump delivering fixed fluid pressure at 10.3 MPa, thermostated extraction cartridge, electrovalve and extract collection device. Ten g of milled bark was packed into a 100 mL extraction cartridge. Both ends of the extraction cartridge were packed with glass wool to fill up void spaces and a cellulose filter was used at the outlet of the extraction cartridge to eliminate particles in the extract. Extraction was performed using single cycle mode with approximately 80 mL of n-hexane as the solvent and 90 s nitrogen purges. Extraction runs were performed in triplicate. Table 2 Factors investigated in the study of the SFE and PLE processes. Level L1

L2

L3

Factors in SFE process A Pressure (bar) B Temperature (◦ C) C Feed particle size (␮m) D Dynamic extraction time (min)

200 50 683 40

300 65 1003 60

400 80 1413 80

Factors in PLE process A Flush volume (%) B Temperature (◦ C) C Feed particle size (␮m) D Static extraction time (min)

60 70 683 5

70 85 1003 10

80 100 1413 15

1 2 3 4 5 6 7 8 9

Variables

Density of CO2 (kg/m3 )

A Pressure (bar)

B Tempera-ture (◦ C)

C Particle size, MMD (␮m)

D Dynamic time (min)

200 200 200 300 300 300 400 400 400

50 65 80 50 65 80 50 65 80

683 1003 1413 1003 1413 683 1413 683 1003

40 60 80 80 40 60 60 80 40

784 691 594 871 809 746 924 874 823

Hildebrand solubility parameter √ ( cal/cc)

ID3 Magnolol yield (%, w/w)

ID4 Mag:hon ratio

ID5 Antioxidant activity (␮L/␮g)

8.275 7.293 6.269 9.189 8.537 7.867 9.748 9.219 8.685

1.353 (0.028) 1.063 (0.293) 1.421 (0.010) 1.705 (0.149) 1.392 (0.142) 1.900 (0.213) 1.368 (0.216) 1.504 (0.301) 2.279 (0.386)

0.024 (0.012) 0.009 (0.003) 0.026 (0.003) 0.026 (0.003) 0.018 (0.006) 0.036 (0.005) 0.024 (0.010) 0.030 (0.010) 0.058 (0.033)

0.548 (0.153) 0.312 (0.059) 0.669 (0.093) 0.480 (0.020) 0.410 (0.062) 0.573 (0.036) 0.454 (0.102) 0.480 (0.106) 0.728 (0.313)

24.267 (6.196) 35.237 (3.731) 25.800 (0.802) 18.464 (1.535) 23.212 (4.455) 15.991 (1.324) 20.261 (3.674) 16.269 (1.824) 13.654 (2.830)

15.125 (4.054) 10.438 (1.166) 12.063 (1.100) 14.882 (6.701) 24.074 (1.604) 23.918 (5.279) 23.916 (11.024) 26.696 (7.748) 26.296 (12.189)

Average outcome

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One gram of milled bark was placed in a cellulose thimble. Extraction was carried out with 100 mL n-hexane for 8 h in a Soxhlet extractor (Model B-811, Buchi, Flawil, Switzerland).

Expt

2.6. Soxhlet extraction

2.7. Determination of extractable solids

PLE and SE extracts were allowed to stand overnight in the refrigerator at 4 ◦ C to allow sedimentation of particles to take place, and further clarified by filtration through Whatman Grade 1 filter paper. SFE, PLE and SE extracts were then dried under reduced pressure at 40 ◦ C using a rotary evaporator (Model N1001S-W, Eyela, Tokyo, Japan). The percentage of extractable solids (ES) was determined gravimetrically as the percentage weight of dry extract with respect to weight of milled material used. ID2 Honokiol yield (%w/w)

Fig. 2. HPLC chromatogram of reference standards. ID1 ES yield (%, w/w)

Table 3 The L9 (3)4 Taguchi design matrix for supercritical fluid extraction of Magnolia officinalis bark and the resultant outcome indices.

Numbers in parentheses indicate standard deviation (n = 3).

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2.8. HPLC analysis The dry extract was reconstituted with a known volume of methanol and passed through a 0.45 ␮m filter (Sartorius, Germany) to remove any particulate matter. HPLC analysis of the samples was conducted using a modified method reported by Tsai and Chen [46]. For all experiments, a HPLC unit (Shimadzu Corp., Japan) equipped with a binary gradient pump, autosampler, column oven and diode array detector (DAD) was used to assay honokiol and magnolol in the extracts. A reversed-phase column (Thermosil® C18, 5 ␮m, 200.0 mm × 4.6 mm I.D., Thermo Electron Co., Pittsburg, PA, USA) fitted with a guard column (UniguardTM HPLC Column Protection System with Drop-in cartridge, Thermosil® C18, 5 ␮m, 10.0 mm × 2.0 mm I.D., Thermo Electron Co., Pittsburg, PA, USA) and isocratic elution using a mobile phase of acetonitrile:0.1% (v/v) phosphoric acid (65:35) of pH 2.3–2.4 at a flow rate of 1.0 mL/min were employed to separate honokiol from magnolol. Detection was performed at wavelengths of 208 nm for honokiol and 203 nm for magnolol at 25 ◦ C, as determined by the max obtained from the DAD. The reference chromatogram is shown in Fig. 2. The sample injection volume was 10 ␮L. The recoveries of magnolol and honokiol from Magnolia bark obtained by SFE, PLE and SE were calculated as the percentage weight of the compound with respect to the milled bark used (%, w/w). The selectivity of magnolol over honokiol was described by the magnolol:honokiol ratio. 2.9. Preparation of reference standards The reference standards, magnolol and honokiol, were accurately weighed and dissolved in methanol to prepare stock standard solutions ranging from 3.5–35 ␮g/mL for magnolol and 1.5–15 ␮g/mL for honokiol. Calibration plots were obtained using these stock solutions. 2.10. Experimental design for SFE and PLE Using the L9 (3)4 Taguchi design matrix, four variables at three levels each were investigated for the SFE and PLE processes (Tables 2–4). The extraction was carried out in triplicate for each set of conditions. Extraction temperatures chosen for PLE were above the boiling point of n-hexane at normal atmospheric pressure (i.e. 69 ◦ C). The effects of the variables on five outcome indices (IDx ), i.e. ES (ID1 ), honokiol recovery (ID2 ), and magnolol recovery (ID3 ); magnolol:honokiol ratio (ID4 ) and antioxidant activity (ID5 ) were determined. The kx,y value, which is the average for each level (Ly ), of a particular variable was calculated. The Rx value, defined by the difference between the maximum and minimum kx,y values for a particular variable and process outcome indicates the influence of the variable, with a greater Rx value corresponding to greater influence. The percent contribution (PCx ), which is computed from the analysis of variance (ANOVA) quantifies the influence of the variable [16]. The percent contribution of error (PCerror ) is defined as the remaining percentage not accountable by the tested variables and takes into account natural variances of the starting material. 2.11. Assay of antioxidant activity The free radical scavenging activity of the extracts was determined by the 2,2-diphenyl-1-picryl-hydrazyl (DPPH) assay, where the DPPH reagent served as the free radical. The test concentrations were modified from Marwah et al. [47] while the DPPH scavenging activity was calculated using equation 1, as reported by Maisuthisakul et al. [48]. In this assay, a known amount of dry extract was reconstituted with methanol to prepare a stock

solution. Different amounts of the stock solution were each further diluted with methanol to 1.56 mL and mixed with 40 ␮L of methanolic solution consisting of 2 mM DPPH. Each mixture was placed in a polyethylene microfuge tube and allowed to stand in dim light at ambient temperature of 20 ◦ C for 30 min. It was then subjected to centrifugation (Model 2100, Kubota, Tokyo, Japan) at 1165 × g for 5 min. The supernatant was removed for absorbance reading at 517 nm (Genesys 10 UV, ThermoSpectronic, Madison, WI, USA). Determinations were conducted in triplicates for each sample. DPPH radical scavenging activity (%) =

A0 − (A1 − AS ) × 100 A0

(1)

where A0 is the absorbance of the control solution containing only DPPH, A1 is the absorbance of the test supernatant and As is the absorbance of the corresponding supernatant without DPPH. A plot of the DPPH radical scavenging activity versus concentration of extract was constructed. The IC50 value, which is the concentration of extract corresponding to 50% of A0 , was obtained from the plot. Antioxidant activity was defined as the inverse of the IC50 value. 2.12. Statistical analysis One way analysis of variance (ANOVA) was employed to assess the influence of the variables and to identify the optimal level of setting for each variable. This was carried out using the statistical analysis software, SPSS v.13 (Chicago, IL, USA) with a level of significance set at 0.05.

3. Results and discussion 3.1. Distribution of plant tissues in the different size fractions Typically, different size fractions are obtained by passing the milled material through a nest of sieves and extracting each fraction to determine the influence of particle size on the extraction process. Using this approach, it was found that the composition of the different size fractions of milled Magnolia bark were not similar. Size fractions above 1.7 mm contained mostly fibrous material; those between 1–1.4 mm had large amounts of parenchyma cells, and those below 0.5 mm contained mainly broken cellular material. A similar study by del Valle and Uquiche [49] reported tough, lignified testa fragments in abundance in larger fractions of oil seeds, while brittle, oil-rich germ fragments were concentrated in smaller size fractions. Different plant tissues may exhibit different mass transfer resistances, which would in turn complicate the interpretation of extraction results [50]. Thus, for this study, the bark was milled under different conditions to produce batches of different size fractions. Each batch was then used for extraction. The particle size and particle size distribution, as represented by the span value, for each batch is shown in Table 1.

3.2. Comparison of extracts obtained by SE, SFE and PLE The quality of the SE, PLE and SFE extracts of Magnolia bark differed markedly. SE extracts were dark yellow with only slight fragrance while SFE extracts were bright yellow with strong fragrance. Superior organoleptic quality of SFE extracts was also reported in other studies [51–53]. In contrast, PLE extracts were dark brown and more turbid, indicating co-extraction of more substances. Both PLE and SE extracts contained significant amounts of sediments and had to be filtered prior to HPLC analysis. On the other hand, SFE extracts could be analysed directly post-extraction.

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297

Table 4 The L9 (3)4 Taguchi design matrix for pressurized liquid extraction of Magnolia officinalis bark and the resultant outcome indices. Expt

1 2 3 4 5 6 7 8 9

Average outcome ± standard deviation (n = 3)*

Variables A Flush volume (%)

B Temperature (◦ C)

C Particle size, MMD (␮m)

D Static time (min)

ID1 ES yield (%, w/w)

ID2 Honokiol yield (%, w/w)

ID3 Magnolol yield (%, w/w)

ID4 Mag:hon ratio

ID5 Antioxidant activity (␮L/␮g)

60 60 60 70 70 70 80 80 80

70 85 100 70 85 100 70 85 100

683 1003 1413 1003 1413 683 1413 683 1003

5 10 15 15 5 10 10 15 5

1.716 (0.246) 1.810 (0.143) 2.192 (0.236) 1.547 (0.251) 1.764 (0.174) 2.332 (0.225) 1.618 (0.127) 2.432 (0.200) 1.803 (0.256)

0.037 (0.011) 0.050 (0.007) 0.063 (0.009) 0.032 (0.009) 0.055 (0.014) 0.117 (0.030) 0.045 (0.012) 0.063 (0.020) 0.071 (0.015)

0.476 (0.080) 0.556 (0.064) 0.607 (0.069) 0.433 (0.074) 0.550 (0.107) 1.074 (0.231) 0.495 (0.105) 0.641 (0.152) 0.686 (0.098)

13.144 (1.835) 11.177 (0.258) 9.674 (0.355) 13.692 (1.514) 10.096 (0.667) 9.254 (0.443) 11.001 (0.731) 10.319 (0.808) 9.773 (0.731)

16.984 (2.870) 25.714 (4.949) 21.667 (2.887) 14.335 (1.027) 14.335 (1.027) 14.423 (1.665) 12.288 (0.906) 20.000 (0) 16.389 (3.758)

Numbers in parentheses indicate standard deviation (n = 3).

65 ◦ C, the effect of lower fluid density predominated over the effect of higher solute volatility as indicated by the lower percentage of ES. However, further increase in temperature to 80 ◦ C led to a more predominant vapour pressure effect, thus improving the solubility of more compounds from Magnolia bark and thus higher percentage of ES. Clearly, the influence of temperature on the percentage of ES was governed by the balance between the effects of fluid density and solute volatility. Pressure affected all the extraction outcomes except magnolol recovery. It was found to be the main factor (PC > 50%) affecting the selectivity of magnolol over honokiol. Since magnolol and honokiol are isomers and possess similar molecular weights, the difference in selectivity could be due to other factors such as melting point, matrix–analyte interaction and their relative polarities. Honokiol possesses a lower melting point than magnolol (87.5 ◦ C vs 102 ◦ C) and was expected to be extracted with greater ease as it would have higher volatility in the supercritical CO2 [55]. However, it was not observed in this study. On the contrary, hon-

3.3. Effects of process variables on SFE Supercritical fluids of higher densities have greater solvating power and thus allowed higher specific extraction of bioactives [13,54]. Hence, conditions that corresponded to higher densities of supercritical CO2 were employed. Both temperature and pressure were dominant variables affecting the ES, honokiol and magnolol recoveries, with insignificant contribution by dynamic extraction time (Table 5). Temperature exerted a greater influence than pressure, as indicated by higher PC values for temperature. The effects of the different variables on the extraction outcomes were further analysed using the kx,y and R values (Table 5). In SFE, the density of the supercritical fluid is decreased while the volatility of solute is increased by raising the temperature. A decrease in density of the supercritical CO2 would decrease its extraction capability [10]. In contrast, increase in solute volatility is expected to enhance extraction and increase the percentage of ES. Regardless of the pressure, when temperature was increased from 50 to

Table 5 kx,y and R values for factors investigated in the supercritical fluid extraction of Magnolia officinalis bark. IDx

kx,y and Rx

A Pressure (bar)

B Temperature (◦ C)

1. ES yield (%, w/w)

k1,1 k1,2 k1,3 R1 PC1 (%)

1.279 1.666 1.717 0.438 24.16†

1.475 1.320 1.866 0.547 34.30†

1.586 1.682 1.394 0.289 7.66†

1.675 1.444 1.543 0.231 3.88

30.00

2. Honokiol yield (%, w/w)

k2,1 k2,2 k2,3 R2 PC2 (%)

0.020 0.027 0.037 0.017 14.07†

0.025 0.019 0.040 0.021 23.49†

0.030 0.031 0.023 0.008 0.66

0.033 0.023 0.027 0.011 2.32

59.46

3. Magnolol yield (%, w/w)

k3,1 k3,2 k3,3 R3 PC3 (%)

0.510 0.488 0.554 0.067

0.494 0.401 0.657 0.256 37.79†

0.534 0.507 0.511 0.027

0.562 0.447 0.543 0.115 5.20

57.01

4. Magnolol:honokiol ratio

k4,1 k4,2 k4,3 R4 PC4 (%)

28.435 19.222 16.728 11.707 53.08†

20.997 24.906 18.482 6.424 13.31†

18.842 22.452 23.091 4.249 5.76†

20.377 23.830 20.178 3.652 4.27

23.58

5. Antioxidant activity (␮L/␮g)

k5,1 k5,2 k5,3 R5 PC5 (%)

12.542 20.958 25.636 13.094 37.95†

17.974 20.402 20.759 2.785

21.913 17.205 20.018 4.707

21.832 19.424 17.880 3.951

* †

*

Pooled into error. Indicates that the effect of the parameter was significant at p < 0.05 (ANOVA).

*

C Particle size (␮m)

*

*

D Dynamic extraction time (min)

*

Error

62.05

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okiol recovery was much lower than the corresponding magnolol recovery. Magnolol (log Poctanol/water 4.43, Chemistry Database http://chemdb.niaid.nih.gov/struct search/all/url search.asp?aids no = 002239) is also slightly more lipophilic than 4.15, Chemistry Database honokiol (log Poctanol/water http://chemdb.niaid.nih.gov/struct search/misc/url search.asp?cas no = 35354-74-6). This aptly explains the greater affinity of magnolol for the relatively non-polar supercritical CO2 . The above findings collectively showed that extraction by supercritical CO2 was highly selective and it was mainly governed by the solubility of the compound in the supercritical fluid. Despite its influence on solvating power, higher pressure was found to have insignificant effect on magnolol recovery. The relatively small percent difference (12%) between the maximum (0.554%, w/w) and minimum (0.488%, w/w) recoveries of magnolol showed that the solubility of magnolol had almost reached its limit in the supercritical CO2 . Hence, further increase in solvating power by adjusting the pressure did not offer any significant advantage in the extraction of magnolol. Pressure was also the only significant factor influencing the antioxidant activity of the Magnolia bark extracts (Table 5). As pressure was found to affect the recoveries of honokiol and ES, but not magnolol, the increased antioxidant activity was due to honokiol and other compounds excluding magnolol. This was previously confirmed in our laboratory whereby honokiol was found to possess an antioxidant activity 2.7 times higher than that of magnolol. The markedly higher percent difference for antioxidant activity (104%) compared to honokiol recovery (85%) and percentage of ES (34%) seemed to suggest that the compounds interacted synergistically. More importantly, it was noted that the recovery of magnolol was affected by temperature only while that of honokiol was affected by both temperature and pressure. The influence of particle size was relatively small, accounting for about 8% of the effect on the percentage of ES and no significant effect on honokiol and magnolol recoveries. This could be attributed

to the greater diffusion coefficient and penetrability of supercritical CO2 into the plant matrix compared to liquid solvents. Similar findings whereby influence of particle size and bed packing were inconsequential in SFE were also reported with other matrix types [56–60]. Overall, the findings indicated that extraction of honokiol and magnolol by supercritical CO2 was rapid and the extraction efficiency was controlled mainly by the solubility of these compounds in the supercritical fluid, rather than by internal mass transfer resistance. This has economic significance as plant materials need not be custom-milled nor finely divided, thus reducing production time and cost. Interestingly, particle size was a significant factor (ANOVA, p < 0.05) in the selectivity for magnolol over honokiol (Table 5). Magnolol was reported to be almost 8 times higher in concentration than honokiol in magnolia bark obtained from the Zhejiang province, China [61]. Honokiol was postulated to be retained more tightly in the plant matrix compared to magnolol and was extracted with greater difficulty. For the same feed weight, smaller particles presented higher total surface area per volume for extraction. Hence, an increase in particle size would be expected to result in poorer extraction of honokiol resulting in greater selectivity of magnolol over honokiol. In the extraction of Magnolia bark, it is desirable to obtain high recoveries of magnolol and honokiol while minimizing coextraction of other constituents, as well as to obtain high biological activity of the extract. From the Taguchi design, the optimum extraction conditions to achieve this were derived from the highest kx,y value for each outcome index, IDx. For SFE, the optimum extraction conditions were pressure 40 MPa, temperature 80 ◦ C and dynamic time 40 min. As particle size was not a significant factor influencing magnolol and honokiol recoveries and antioxidant activity of extract, a particle MMD of 1003 ␮m could be chosen to reduce pre-extraction processing time. Thus, the final optimum extraction conditions coincide with SFE experiment 9. As the PCerror for honokiol and magnolol recoveries as well as antioxidant activities were greater than 50% (Table 5), this implied that

Table 6 kx,y and R values for factors investigated in the pressurized liquid extraction of Magnolia officinalis bark. IDx

kx,y and Rx

A Flush volume (%)

B Temperature (◦ C)

C Particle size (␮m)

D Static extraction time (min)

1.627 2.002 2.109 0.482 32.58†

2.160 1.720 1.858 0.440 25.24†

1.761 1.920 2.057 0.296 9.48†

32.70

Error

1. ES yield (%, w/w)

k1,1 k1,2 k1,3 R1 PC1 (%)

1.906 1.881 1.951 0.070

2. Honokiol yield (%, w/w)

k2,1 k2,2 k2,3 R2 PC2 (%)

0.050 0.068 0.060 0.018 4.95

0.038 0.056 0.084 0.045 45.60†

0.072 0.051 0.054 0.021 9.77†

0.054 0.071 0.053 0.018 6.62

33.06

3. Magnolol yield (%, w/w)

k3,1 k3,2 k3,3 R3 PC3 (%)

0.546 0.685 0.607 0.138 5.09

0.468 0.581 0.789 0.321 39.60†

0.730 0.558 0.549 0.181 13.99†

0.569 0.708 0.560 0.148 8.35†

32.97

4. Magnolol:honokiol ratio

k4,1 k4,2 k4,3 R4 PC4 (%)

11.332 11.014 10.364 0.967 3.31

12.612 10.531 9.567 3.046 55.92†

10.906 11.547 10.257 1.290 7.48†

11.004 10.477 11.228 90.576

k5,1 k5,2 k5,3 R5 PC5 (%)

21.455 14.364 16.225 7.091 40.38†

14.535 20.016 17.493 5.481 21.43†

17.136 18.813 16.096 2.716 3.57

15.903 17.475 18.667 2.765 3.71

5. Antioxidant activity (␮L/␮g)

* †

*

Pooled into error. indicates that the effect of the parameter was significant at p < 0.05 (ANOVA).

*

33.29

59.62

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not all variables that affected the extraction process were taken into account, or compounds in addition to honokiol and magnolol contributed to the antioxidant activity of the extracts. Nevertheless, the pressure and temperature were important factors affecting the extraction process. 3.4. Effect of process variables on PLE n-Hexane was chosen as the solvent for both PLE and SE processes as a previous study showed that it produced appreciable recoveries with significantly lower content of extraneous compounds. The selection of n-hexane also enabled the matching of the Hildebrand solubility parameter of the solvent to that of supercritical CO2 . This allowed a common baseline for comparing the mechanistic differences among the different extraction processes. The influence of the PLE variables on the percentage of ES, honokiol and magnolol recoveries was found to decrease as follows: temperature > particle size > static extraction time (Table 6). Flush volume had insignificant effect on PLE. PCerror for percentage of ES and recoveries of target compounds was around 30% and this was postulated to be due to natural variations in the starting material. An increase in extraction temperature from 70 to 100 ◦ C led to increased recoveries but reduced selectivity of magnolol over honokiol (Table 6). Purity of the extract was also compromised due to greater co-extraction of extraneous material, as indicated by the greater increase in percentage of ES compared to the recoveries of magnolol and honokiol. The recoveries of honokiol and magnolol increased with temperature, indicating that the stability of these compounds was not compromised by the temperature employed. However, the antioxidant activity of the extracts was found to be diminished when temperature was increased to 100 ◦ C, implying deterioration of certain components that were synergistic with honokiol and magnolol or extraction of certain components that were antagonistic. Particle size exerted a significant influence on the performance of PLE but not SFE. This was a significant finding which indicated that supercritical fluids had greater penetrative ability than highly pressurized liquids. The smallest particle size generated the greatest percentage of ES, and honokiol and magnolol recoveries, which could be ascribed to the larger total surface area presented by smaller particles for extraction. In PLE, the static extraction mode is when a fixed volume of solvent is used to extract the extraction bed over a fixed period of time (i.e. the static extraction time) [62]. The percentage of ES was found to increase with static extraction time, but the recoveries of magnolol and honokiol decreased when static extraction time was longer than 10 min. As exposure to high temperatures did not compromise the stability of magnolol and honokiol, the increased static extraction time could have resulted in readsorption of both compounds onto the botanical matrix thus reducing their recovery in the extract [51–52,63]. It was also possible that the decreased recoveries of the compounds were due to the adsorption of the compounds to the extractable solids. Flush volume had no significant effect on quantitative recoveries, which is in agreement with the extraction of active compounds from Dictamnus dasycarpus root cortex [63]. Larger flush volumes were associated with greater loss in antioxidant activity. This could be due to degradation of the active principles during the post-extraction evaporation process

299

whereby larger volumes took a longer time to evaporate, resulting in longer exposure of the extract to heat. From the Taguchi design, the optimum PLE conditions for maximal recoveries of honokiol and magnolol from Magnolia bark were flush volume 70%, temperature 100 ◦ C, particle MMD 683 ␮m and static extraction time 10 min, coinciding with PLE experiment 6. However, the flush volume and temperature should be modified to 60% and 85 ◦ C respectively, to obtain maximum antioxidant activity. It was not necessary to modify particle size and extraction time as they did not exert significant influence on antioxidant activity. This again implied that other compounds present in the extract act synergistically with honokiol and magnolol to result in greater antioxidant activity even though recoveries of both of these compounds were low. 3.5. Comparison of SFE and PLE with conventional Soxhlet extraction There was no significant difference (ANOVA, p > 0.05) in the respective recoveries obtained by SE from different particle sizes of milled Magnolia bark, indicating that this method of extraction is exhaustive (Table 7). In this study, the Hildebrand solubility parameter was used to match the solvency behaviour of the supercritical fluid to that of n-hexane in SE. At SFE conditions matching the Hildebrand solubility parameter of n-hexane, i.e. SFE experiment 2, the percentage of ES, honokiol and magnolol recoveries were much lower than those obtained by PLE and SE. Thus, the ability to predict the extraction of honokiol and magnolol from Magnolia bark using the Hildebrand solubility parameter was limited in the extraction of herbs compared to other applications [64,65]. Strong interaction between plant tissues and compounds (i.e. matrix–analyte interaction) is reported to reduce extraction yield [10]. Therefore, the results in this study clearly showed that although supercritical fluids were highly diffusive and penetrative, they were less able than liquids in disrupting the matrix–analyte interaction. Furthermore, the solubility of a compound is expected to be higher in a liquid than in a supercritical fluid as the viscosity of the latter is usually an order of magnitude lower than that of a liquid [66]. At the optimal conditions of SFE, however, the percentage of ES obtained was 98% and 62% of the ES obtained at the optimal conditions of PLE and SE respectively. The corresponding honokiol recovery was 50% while the magnolol recovery was 68% of those obtained by PLE at the above conditions; and 52% and 67% of magnolol and honokiol recoveries respectively obtained by SE. The relationship between percentage of ES and particle size was similar for both SFE and SE, with the intermediate particle size demonstrating the highest ES. Reduced percentage of ES at larger particle sizes were explained by the increased mass transfer resistance. Reduction in percentage of ES with the smallest particle size was most likely due to loss of volatile compounds as the surface area increased [67,68]. This was not observed in PLE due to the relatively shorter extraction times. At the optimal conditions of PLE, the honokiol and magnolol recoveries were comparable to those obtained from SE (p > 0.05) even though percentage of ES obtained from PLE was significantly lower (p < 0.05). The proportions of active principles in the extracts obtained by PLE were higher compared to SE (honokiol 5% vs 3%; magnolol 46% vs 30%). On the other hand, the SFE and SE extracts had comparable proportions of the active principles, which were lower than those obtained at the optimal PLE

Table 7 Comparison of extracts obtained by Soxhlet extraction of Magnolia officinalis bark.

Size 1 Size 2 Size 3

ES yield (%, w/w)

Honokiol yield (%, w/w)

Magnolol yield (%, w/w)

Magnolol:honokiol ratio

Antioxidant activity (␮L/␮g)

2.908 (0.343) 3.694 (0.537) 2.764 (0.246)

0.090 (0.017) 0.112 (0.021) 0.081 (0.011)

1.014 (0.056) 1.090 (0.158) 0.881 (0.051)

11.68 (3.15) 9.84 (0.53) 11.05 (1.81)

23.107 (1.033) 14.081 (2.372) 17.820 (1.079)

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conditions. This was attributed shorter PLE time for co-extraction of other substances. Clearly, PLE is a more efficient alternative to SE. The magnolol:honokiol ratio could be modified to a great extent by varying the operation conditions of SFE. This property of SFE is especially important in the isolation of closely related bioisomers, which are typically found in plants. More importantly, the antioxidant activity of the SFE extract was higher than that of PLE and SE extracts obtained at optimal conditions. This was attributed to lower operational temperature which provided gentler extraction suitable for thermolabile antioxidant compounds. The results are in agreement with findings reporting similar or greater antioxidant activity of SFE extracts compared to those obtained by the traditional method [69–72]. 4. Conclusions The performance of PLE is significantly affected by temperature, particle size and static extraction time but not flush volume. It is a more economical alternative to SE in exhaustive extraction as it requires less solvent and time. On the other hand, the performance of SFE is significantly affected by temperature and pressure, and to a smaller extent, particle size. Supercritical fluids have better penetrative ability than highly pressurized liquid solvents, hence energy is conserved as grinding the plant material to a suitable particle size is not as crucial in SFE as in PLE. However, the extraction efficiency of honokiol and magnolol using SFE is limited by its solubility in supercritical CO2 . The selectivity of SFE can be manipulated by varying the operation conditions. The extracts obtained by SFE also possess stronger antioxidant activity compared to those obtained by PLE and SE using n-hexane as solvent. In addition, SFE results in solvent-free extracts thus amounting to time savings for post-extraction clean up. Further studies should be aimed at improving the recoveries of honokiol and magnolol by addition of a polar modifier to disrupt matrix–analyte interactions. Conflicts of interest The authors declare no conflict of interest. Acknowledgements This work was funded by the Academic Research Fund (R148-000-055-112) and by GEA-NUS Pharmaceutical Processing Research Laboratory, National University of Singapore. Appreciation is extended to Prof. Baolin Guo, Herbarium of Institute of Medicinal Plant Development (IMD), Chinese Academy of Medical Sciences, Beijing, China for identification of plant material. Thanks are also extended to Dr. Cai Bin and Dionex Corp., for generous loan of the ASE 100 equipment. Ms Emily Cheah LC is a recipient of the postgraduate research scholarship from National University of Singapore. References [1] B. Benthin, H. Danz, M. Hamburger, J. Chromatogr. A 837 (1999) 211–219. [2] J. Chen, W. Li, B. Yang, X. Guo, F.S.-C. Lee, X. Wang, Anal. Chim. Acta 596 (2007) 273–280. [3] A.L. Dawidowicz, D. Wianowska, J. Pharm. Biomed. Anal. 37 (2005) 1155–1159. [4] E.-S. Ong, S.-O. Woo, Y.-L. Yong, J. Chromatogr. A 904 (2000) 57–64. [5] A. Sae-Yun, C. Ovatlarnporn, A. Itharat, R. Wiwattanapatapee, J. Chromatogr. A 1125 (2006) 172–176. [6] Dionex Corp., ASE 100 Accelerated Solvent Extractor Data Sheet, California, USA. [7] R. Carabias-Martínez, E. Rodríguez-Gonzalo, P. Revilla-Ruiz, J. HernándezMéndez, J. Chromatogr. A 1089 (2005) 1–17. [8] A.T.W. Eng, M.Y. Heng, E.S. Ong, Anal. Chim. Acta 583 (2007) 289–295. ˜ [9] J.A. Mendiola, M. Herrero, A. Cifuentes, E. Ibanez, J. Chromatogr. A 1152 (2007) 234–246.

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