Article pubs.acs.org/IECR
Development of Circulating Ultrasounic-Assisted Online Extraction Coupled to Countercurrent Chromatography and Centrifugal Partition Chromatography for Simultaneous Extraction and Isolation of Phytochemicals: Application to Ligusticum chuanxiong Hort Yuchi Zhang,† Chunming Liu,*,† Yanjuan Qi,† Yuchun Li,‡ and Sainan Li§ †
Central Laboratory, Changchun Normal University, No. 677 North Changji Road, Erdao District, Changchun 130032, China Traditional Chinese Medicine Academy of Science of Jilin Province, No. 1745 Gongnong Road, Chaoyang District, Changchun 130021, China § Faculty of Chemistry, Northeast Normal University, No. 5268 Renmin Street, Nanguan District, Changchun 130024, China ‡
S Supporting Information *
ABSTRACT: Circulating ultrasonic-assisted extraction (CUAE) was developed as a novel method for extraction of medicinal herbs, and the method was validated. In addition, the novel hyphenated technique comprising CUAE coupled with countercurrent chromatography (CCC) and centrifugal partition chromatography (CPC) was developed and applied to the continuous extraction and online isolation of chemical constituents from Ligusticum chuanxiong Hort. The optimum extraction parameters, including the extraction time of 30 min, extraction temperature of 45 °C, ultrasound power of 300 W, and the liquid−solid ratio of 10 mL/g were determined by response surface methodology. Furthermore, a schematic and the mechanism of online CUAE coupled with CCC and CPC were presented. Three lactones, levistolide A (52.2 mg), Z-ligustilide (48.3 mg), and wallichilide (118.2 mg), with respective purities of 95.8, 96.7, and 96.2%, were obtained from 500 g of the L. chuanxiong raw material using CUAE/CCC. In contrast, senkyunolide A (26.2 mg), levistolide A (34.2 mg), and wallichilide (95.1 mg), with respective purities of 96.2, 95.3, and 96.1%, were obtained from 500 g of the L. chuanxiong raw material by using CUAE/CPC employing the two-phase solvent system comprising n-hexane−ethyl acetate−methanol−water in a volume ratio of 4:3:4:2 (v/v). Compared with reference extraction methods, scientific and systematic extraction and isolation of natural products was achieved with the instrumental setup, and this system has great prospects for industrial application, where the combined use of CCC and CPC can enhance the separation efficiency.
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INTRODUCTION The root of Ligusticum chuanxiong Hort. (chuanxiong) is traditionally used in Chinese medicine for treating headaches,1 ischemic stroke,2 anemia,3 and cerebral vascular disease.4 The major active ingredients of L. chuanxiong are lactones. Studies have shown that lactones such as senkyunolide A and Zligustilide can inhibit platelet aggregation and relax the uterus, trachea, and vascular smooth muscle. These compounds can also be used to treat coughs, menstrual disorder, and hypertension.5 The chemical structures of some of the compounds obtained from L. chuanxiong are shown in Figure 1. Sample preparation is a crucial step in the analysis of medicinal herbs. Extraction of the desired chemical components of the herbal material is a prerequisite for further isolation and characterization. In conventional methods, the extraction of medicinal herbs is accomplished by heating, boiling, refluxing, or using other thermal techniques. These methods, however, result in the loss of desired components because of ionization, hydrolysis, and oxidation of the compounds during extraction.6,7 In addition, the thermal techniques require large amounts of solvent and long extraction times.8−10 Therefore, development of new sample preparation techniques with the capacity for extraction of most of the useful constituents of medicinal plants in less time at low cost is highly desirable.11 © 2015 American Chemical Society
The modern techniques based on ultrasound-assisted extraction (UAE) are promising alternatives for retrieval of bioactive components from plants.12−14 Some of the major benefits of UAE include enhancement of the extraction yield or extraction rate, incorporation of alternative solvents, and compatibility with heat-sensitive compounds. Furthermore, ultrasound has the unique capacity to enhance extraction while simultaneously encapsulating the extracted substance in the extraction fluid by hydroxyl radical and microsphere formation.15 UAE is usually performed in an ultrasonic cleaning bath. It is possible to obtain good extraction yield with this equipment.16,17 UAE has also been carried out in a glass vessel with a stirrer and a cooling jacket.18 The chemical constituents of fresh as well as dry plant materials can be effectively extracted by UAE. In the case of fresh plant material, the solvent diffuses through the cell wall and leaches out the cell contents once the wall is broken. Ultrasonic irradiation plays a significant role in both steps. For the dried materials, steeping the raw materials Received: Revised: Accepted: Published: 3009
October 23, 2014 March 9, 2015 March 10, 2015 March 10, 2015 DOI: 10.1021/ie504179r Ind. Eng. Chem. Res. 2015, 54, 3009−3017
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Industrial & Engineering Chemistry Research
extraction solution. After CUAE extraction, the extraction solution is pumped into the sample loop, and then pumped into the CCC column. During the CCC separation, the extraction of the medicinal herbs by CUAE is still operative; the extraction solution is then separated by CPC. After CPC separation, the extraction solution is then separated by CCC, and so on. Using this instrumental setup, automated extraction and isolation of natural medicines is achieved, and the integrated system has great prospects for industrial application.
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MATERIALS AND METHODS Reagents and Raw Material. The ethyl acetate, n-hexane, and methanol used herein were analytical-grade (Beijing Chemicals, Beijing, China). Water was purified on a Milli-Q water purification system (Millipore, Boston, USA). Acetonitrile and phosphoric acid were HPLC-grade (Fisher Scientific, Shanghai, China). Ligusticum chuanxiong Hort. was purchased from TongRenTang Medicinal Store and identified by Dr. Yuchi Zhang (Changchun Normal University, Changchun, China). The authentic standards were purchased from the National Institute for Food and Drug Control of China. Apparatus. Circulating ultrasound-assisted dynamic extraction (TGCXZ-10B, Hongxianglong, Beijing, China) was performed with a 10 L extractor comprising a spiral agitator, ultrasound probe, feeding throat (inner diameter (ID) = 10.0 cm), and discharge port (diameter = 6.0 cm). The power of the stirring motor was 200 W; the ultrasonic frequency was 59 kHz. The maximum ultrasonic power was 500 W; the maximum heating power was 1000 W. The rotational speed of the spiral stirrer ranged from 0−1500 rpm. The length and diameter of the ultrasound probe were 10 and 3 cm, respectively. The CCC system was reequipped with the commercial TAUTO TBE− 300B Spectrum HSCCC (TAUTO Co., Ltd., Shanghai, China) product with three multilayer coil separation columns connected in series (ID of the tubing = 1.5 mm, total volume = 300 mL). The revolution radius was 5 cm, and the values of β (= r/R, where r is the rotation radius or the distance from the coil to the holder shaft and R is the revolution radius or the distance between the holder axis and central axis of the centrifuge) of the multilayer coil varied from 0.5 at the internal terminal to 0.8 at the external terminal. The revolution speed of the apparatus could be regulated within the range of 0 to 1000 rpm using a speed controller; 850 rpm was used in the present study. CPC was performed on a SIC CPC-240 (Kyoto, Japan) instrument that was modified in our laboratory. The CPC column is a stacked circular partition disk rotor that contains 2136 partition cells with a total internal volume of about 240 mL. The column was connected to the injector and the detector through two high-pressure rotary seals. A four-port valve, integrated in the CPC apparatus, facilitated operation in either the ascending or the descending mode depending on the relative density of the mobile and stationary phases. Ultrahighperformance liquid chromatography (UHPLC) was carried out on a Waters Acquity UPLC−H class (Milford, MA, USA) instrument. Electrospray ionization mass spectrometry (ESI− MS) was performed on an LCQ FLEET ion-trap mass spectrometer (Thermo Finnigan, San Jose, CA, USA). Nuclear magnetic resonance (NMR) spectra were recorded using a Bruker AV-500 spectrometer (Bruker BioSpin, Rheinstetten, Germany). Measurement of Partition Coefficient Values. The composition of the two-phase solvent system was selected on the basis of the partition coefficient (K) of the target
Figure 1. Structures of target compounds from the leaves of Ligusticum chuanxiong Hort.
in solvent facilitates swelling and hydration and mass transfer of the soluble constituents of the material to the solvent by diffusion and osmotic processes.19−21 Both cases utilize static and/or semidynamic extraction processes because of which the chemical constituents of the raw material become saturated in the extraction solution. Therefore, it is necessary to develop a dynamic UAE protocol to prevent saturation of the chemical constituents in the extraction solution. Countercurrent chromatography (CCC) and centrifugal partition chromatography (CPC) are well-developed and widely used support-free liquid−liquid partition chromatographic techniques. A major advantage of both of these techniques over conventional column chromatography arises from the elimination of irreversible adsorption of the sample onto the solid support.22−26 CCC and CPC differ in terms of the hydrodynamic and hydrostatic mechanisms. Because CCC and CPC operate on the basis of different principles, the compounds separated by CCC and CPC using the same set of two-phase solvent systems are usually different. Therefore, the combined use of CCC and CPC may result in enhanced separation efficiency. Furthermore, because CCC and CPC both utilize a liquid stationary phase, it is possible to combine CUAE with CCC and CPC for extraction and online isolation of the chemical constituents of plant materials. Herein, we present the development of a novel, rapid instrumental setup coupling CUAE to CCC and CPC (CUAE/ CCC/CPC) for the continuous extraction and online isolation of chemical constituents from raw plant material (Figure 1). The stationary phase of CCC and CPC is also used as the 3010
DOI: 10.1021/ie504179r Ind. Eng. Chem. Res. 2015, 54, 3009−3017
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Industrial & Engineering Chemistry Research
Figure 2. Schematic of instrumental setup used to combine CUAE with CCC and CPC.
into the 25 mL sample loop via a six-way valve (ports 1 and 6 were connected). After loading, the six-way valve was turned to connect ports 1 and 2 to flush the extraction solution in the sample loop with the stationary phase in the stationary phase reservoir, followed by pumping the extracted solution into the CCC via the ten-way valve (ports 1/10, 2/3, 4/5, 6/7, and 8/9 were connected). The six-way valve was turned to connect ports 1/6, 2/3, and 4/5. The column of CCC was then rotated at 800 rpm. The ten-way valve was turned to connect ports 1/2, 3/4, 5/6, 7/8, and 9/10, and the mobile phase was pumped into the CCC at a flow-rate of 2.0 mL/min to separate the target compounds. The temperature of the apparatus was set at 40 °C. Peak fractions were collected according to the elution profile and evaporated using a rotary evaporator. During the CCC separation, the stationary phase in the stationary phase reservoir was pumped into the CPC column via the six-way valve (ports 1/6, 2/3, and 4/5 were connected) and the tenway valve (ports 1/2, 3/4, 5/6, 7/8, and 9/10 were connected). The six-way valve was then turned to connect ports 1 and 2 to allow the extraction solution in the 25 mL sample loop to be pumped into the CPC column. The CPC column was allowed to rotate; the ten-way valve was turned to connect ports 1/10, 2/3, 4/5, 6/7, and 8/9. Then, the mobile phase was pumped into the CPC to separate the target compounds. After CPC separation, the CCC column was filled with the extraction solution from CUAE as the stationary phase, and the second stage of CCC/CPC separation was then carried out. By means of the continuous extraction and isolation procedure mentioned above, industrialized separation of chemical constituents from medicinal herbs can be achieved. UHPLC Analysis. The CUAE extracts and each CCC and CPC peak fraction were analyzed by UHPLC. The analysis was performed with a UPLC BEH C18 reverse-phase column (100 mm × 2.1 mm ID, 1.7 μm, Waters, USA) at a temperature of 30 °C; mobile phases: A, acetonitrile and B, 0.5% phosphoric acid. Linear gradients: 0−2 min, 5% A; 2−17 min, 5−100% A; flow rate, 0.4 mL/min, acquisition wavelength, 280 nm. Identification of the CCC and CPC peak fractions was performed by MS and NMR techniques. Optimization of CUAE by RSM. To evaluate the interaction among the numerous factors, the operating conditions were optimized by response surface methodology (RSM) using Box−Benhnken (BBD) data processing software. The factors were applied and optimized with the aid of Design-
compounds in the crude sample. Approximately 3 mg of the crude sample was weighed into a test tube, to which 2 mL of each phase of the equilibrated two-phase solvent system was added. The tube was shaken vigorously to equilibrate the sample thoroughly between the two phases. The respective phases were then separated and evaporated to dryness under N2 gas. The residue was dissolved in methanol and analyzed by HPLC. The partition coefficient (K) is expressed as the peak area of the target compound in the upper phase divided by that in the lower phase. Online CUAE Coupled with CCC and CPC Separation. The instrumental setup of the circulating ultrasonic-assisted extraction (CUAE) coupled with CCC and CPC system is shown in Figure 2. The setup comprises a solvent configuration system, solvent separator, a CUAE, six-port valve, a reservoir for filling the stationary phase of CCC and CPC into the system, a reservoir for filling the mobile phase of CCC and CPC into the system, a ten-port valve, a CCC system, and a CPC system. Each component was connected to the other by pipelines and/or pumps. The stationary phase of CCC and CPC was also used as the extraction solvent for CUAE. First, we optimized the solvent system on the basis of the partition coefficients (K) of the target compounds. Subsequently, the compositions and volume ratios of the reagents for CCC, CPC, and CUAE were determined. Each reagent, including n-hexane, ethyl acetate, methanol, and water, was pumped into the mixer at different flow rates (upper left corner of Figure 2). The mixed and degassed solvents were introduced into the solvent separator, and the mixed solvent was separated into two layers in the solvent separator. The upper organic phase in the solvent separator was pumped into the stationary phase reservoir, whereas the lower aqueous phase in the solvent separator was pumped into the mobile phase reservoir. (The upper organic phase was used as the stationary phase for CCC and CPC; the lower aqueous phase was used as the mobile phase for CCC and CPC.) The raw herbal material was added to the CUAE through the feeding throat. The valve at the top of the solvent separator was turned, and the upper organic phase in the solvent separator was then pumped into the CUE. (The stationary phase of CCC and CPC was used as the extraction solvent.) The spiral agitator in the CUE was then allowed to rotate. During the extraction, the stationary phase of CCC in the stationary phase reservoir was pumped into the CCC column. After extraction, the extraction solution was pumped 3011
DOI: 10.1021/ie504179r Ind. Eng. Chem. Res. 2015, 54, 3009−3017
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Table 1. Partition Coefficients of Senkyunolide A, Levistolide A, Z-Ligustilide, and Wallichilide in Different Solvent Systemsa K values solvent system (volume ratios, v:v:v:v) n-hexane−ethyl n-hexane−ethyl n-hexane−ethyl n-hexane−ethyl n-hexane−ethyl n-hexane−ethyl n-hexane−ethyl n-hexane−ethyl n-hexane−ethyl n-hexane−ethyl
acetate−methanol−water acetate−methanol−water acetate−methanol−water acetate−methanol−water acetate−methanol−water acetate−methanol−water acetate−methanol−water acetate−methanol−water acetate−methanol−water acetate−methanol−water
(5:1:5:1) (5:2:5:1) (5:3:5:1) (3:4:4:3) (3.5:4:4:3) (4:3.5:4:2.5) (4:4:4:2.5) (4:3.5:4:2) (4:3:4:2) (4:3:4:3)
compd 1
compd 2
compd 3
0.20 0.23 0.16 3.31 2.32 1.21 1.48 1.10 0.64 0.62
0.29 0.49 0.29 3.45 2.50 3.41 4.80 1.90 1.36 1.24
0.41 0.45 0.42 7.00 5.51 3.37 4.35 2.70 1.63 1.46
compd 4 b
1.90 2.55 2.10 1.92
a
Compounds: 1, senkyunolide A; 2, levistolide A; 3, Z-ligustilide; and 4, wallichilide. bCompound dissolves completely in the upper or lower phase and dissolve partially in the other phase.
solid ratio, the yields of the target compounds (senkyunolide A, levistolide A, Z-ligustilide, and wallichilide) were employed as the research indicator; the extracts and standard compounds were analyzed by UPLC. The peak areas of senkyunolide A (0.20 mg/mL), levistolide A (0.22 mg/mL), Z-ligustilide (0.20 mg/mL), and wallichilide (0.22 mg/mL) were obtained at different injection volumes (1−10 μL). The regression equations for the standards are presented in Table 2. Good linear relationships were obtained for each compound. RSM was also utilized herein. The extraction results are shown in Table 3, and the ANOVA results are shown in Table 4. In this table, the extraction time, temperature, power, and liquid−solid ratio are defined as variables. The model F value of 26.52 indicated that the model was significant, and there is only a 0.20% chance that a model F value of this size could occur because of statistical noise. The F values of the temperature, power, and liquid−solid ratio were 79.09, 34.08, and 231.65 respectively, which were all higher than 26.52. Values of Prob > F less than 0.0500 indicate that the model terms are significant. The Prob > F values for the extraction temperature, ultrasound power, and liquid−solid ratio were all less than 0.0001, indicating that the model terms were significant. Therefore, the extraction temperature, ultrasound power, and liquid−solid ratio were significant model terms, but the extraction time was not a significant model term. The significant lack of fit was unfavorable despite hopes that the model would fit. The negative predicted R2 implied that the overall mean was a better predictor. This model could be used to navigate the design space. The final equation in terms of coded factors is represented as
Expert 7.0 software. The selected boundary conditions were as follows: 20−60 min extraction time, 30−70 °C extraction temperature, 300−500 W ultrasound power, and 6−10 liquid− solid ratio (mL/g). One-way ANOVA was used to determine if the differences in the extraction yields were significant. The results of UHPLC analysis are expressed as mean ± standard deviation (SD).
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RESULTS AND DISCUSSION CCC, CPC, and CUAE Solvent Optimization. Selection of the two-phase solvent system is the most important step for separation of the analytes by CCC and CPC.27,28 To select a two-phase solvent system for optimizing separation, certain factors must be considered: the retention of the stationary phase should be satisfactory, the set time of the two-phase solvent system should be shorter than 30 s, and the partition coefficient (K) of the target compound should be satisfactory (usually between 0.5 and 2).29,30 Generally, target compounds with lower K values are not well separated, whereas band broadening tends to occur for compounds with higher K values.31 The solvent system used in this study consisted of n-hexane− ethyl acetate−methanol−water in varying volume ratios. The K values of senkyunolide A, levistolide A, Z-ligustilide, and wallichilide in different solvent systems are shown in Table 1. The results indicated that the compounds had low K values in the solvent system comprising n-hexane−ethyl acetate− methanol−water at volume ratios of 5:1:5:1, 5:2:5:1, and 5:3:5:1. Relatively larger K values were obtained with this solvent system at volume ratios of 3:4:4:3 and 3.5:4:4:3. However, the K values of senkyunolide A and levistolide A were very close, which would make them difficult to separate. Levistolide A and Z-ligustilide could not be adequately separated by the solvent system at a volume ratio of 4:3.5:4:2.5 because of the same reason. The solvent system comprising n-hexane−ethyl acetate−methanol−water at a volume ratio of 4:4:4:2.5 was not suitable for separation of the lactones by CCC because the high K value led to a long elution time. Suitable K values were obtained with the solvent system at volume ratios of 4:3:4:2 and 4:3:4:3; the final volume ratio of each reagent in the two-phase solvent system was optimized on the basis of the CCC and CPC chromatograms. Optimization of Senkyunolide A, Levistolide A, ZLigustilide, and Wallichilide Extraction using BBD. To optimize the extraction parameters, including the extraction time, extraction temperature, ultrasound power, and liquid−
Y = 4.82 + 0.22A + 0.60B + 0.39C + 1.03D − 0.000AB + 0.085AC − 0.089AD − 0.21BC + 0.13BD − 0.035CD + 0.18A2 + 0.014B2 − 0.19C 2 + 0.057D2
(1)
where A is the extraction time, B is the extraction temperature, C is the ultrasound power, D is the liquid−solid ratio. The final total extraction yield (Y) of senkyunolide A, levistolide A, Zligustilide, and wallichilide is expressed as (final equation in terms of actual factors) 3012
DOI: 10.1021/ie504179r Ind. Eng. Chem. Res. 2015, 54, 3009−3017
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Table 3. Results of Response Surface Methodology Designa
Y: UPLC peak area of the target compounds. X: Concentrations of the target compounds (μg/mL).
0.9998
no.
A (time, min)
B (temperature, °C)
C (power, W)
D (liquid− solid ratio)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29
40 60 20 60 40 20 40 40 40 60 40 40 40 40 40 20 40 60 60 20 40 60 40 20 40 40 40 40 20
45 45 45 30 45 45 60 45 30 60 60 45 45 45 45 45 30 45 45 30 60 45 45 45 30 45 60 30 60
400 300 500 400 400 400 500 300 500 400 400 400 500 300 400 400 300 400 500 400 300 400 400 300 400 500 400 400 400
15 15 15 15 15 20 15 20 15 15 20 15 10 10 15 10 15 20 15 15 15 10 15 15 20 20 10 10 15
a
yield ± SD (mg/10 g) 4.75 4.24 5.21 4.67 4.85 5.75 5.46 5.45 4.56 5.95 6.45 4.62 3.95 3.05 4.92 3.62 3.45 6.35 5.26 4.02 5.21 4.58 4.96 4.53 5.21 6.21 4.32 3.59 5.30
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
0.08 0.04 0.06 0.08 0.08 0.04 0.06 0.10 0.06 0.07 0.11 0.07 0.08 0.09 0.06 0.06 0.07 0.07 0.08 0.08 0.07 0.09 0.06 0.08 0.07 0.09 0.09 0.07 0.07
n=3
Y = − 5.42187 − 0.027958A + 0.066194B + 0.024717C + 0.12358D + 0.00000AB + 0.0000425AC − 0.0008875AD − 0.000143333BC + 0.0017BD − 0.00007CD + 0.000439583A2 + 0.000062037B2 − 0.0000187292C 2 + 0.00228333D2
(2)
The credibility analysis of the regression equations is summarized in Table 4. The response surfaces for the effect of the independent variables on the total extraction yield of senkyunolide A, levistolide A, Z-ligustilide, and wallichilide are shown in Figure 3. Figure 3a shows the 3D response surface plots at various extraction times (A) and extraction temperatures (B) with a fixed ultrasound power and liquid−solid ratio (0 level). In conjunction with the extraction temperature, the extraction time had little effect on the extraction yield, and the yields of the target compounds increased slightly as the extraction temperature increased. Figure 3b shows the 3D response surface plots with the use of various extraction times (A) and ultrasound powers (C) at a fixed extraction temperature and liquid−solid ratio (0 level). In the combination of the extraction time and the ultrasound power, neither the extraction time nor the ultrasound power had a significant effect on the yields, and the yields of the target compounds increased as the extraction time and the ultrasound power increased. Figure 3c shows the 3D response surface plots at various extraction times (A) and the liquid−solid ratios (D) at a fixed extraction temperature and ultrasound power (0
a
0.20−2.00 wallichilide
Z-ligustilide
levistolide A
senkyunolide A
+
193 [M + H] , 215 [M + Na]+ 381 [M + H]+, 403 [M + Na]+, 191 191 [M + H]+, 381 [2M + H]+, 403 [2M + Na]+ 191, 413 [M + H]+, 435 [M + Na]+
413 → 223, 191
Y = 1.985 × 104X + 1.895 × 103
0.9994 0.20−2.00 Y = 2.364 × 104X + 5.654 × 102 191 → 173 [M − H2O + H]+, 163 [M − C2H4 + H]+, 149 [M − C3H6 + H]+, 145 [M − H2O − CO + H]+, 135 [M − C4H8 + H]+, 117 [M − H2O − CO − C2H4 + H]+, 105 [M − H2O − CO − C3H4 + H]+, 91
0.9996 0.20−2.00 Y = 2.412 × 104X − 2.123 × 103 381 → 191
0.9995 0.20−2.00 Y = 1.536 × 10 X − 8.254 × 10 193 → 175 [M − H2O + H] , 137 [M − C4H8 + H] , 105 [M − H2O − CO − C3H6 + H] , 147, 91
4 +
MS/MS data (m/z)
+
compounds
MS data in positive ion mode (m/z)
Table 2. Analytical Data for Senkyunolide A, Levistolide A, Z-Ligustilide, and Wallichilide
+
regression equation
a
2
linear range of regression equation (μg)
R2
Industrial & Engineering Chemistry Research
3013
DOI: 10.1021/ie504179r Ind. Eng. Chem. Res. 2015, 54, 3009−3017
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solid ratio increased. Figure 3d shows the 3D response surface plots at various extraction temperatures (B) and extraction powers (C), where the other two other parameters are fixed (0 level). The results indicated that the feeding speed of the raw material and the extraction temperature both affected the yields. The yields of the target compounds increased with an increase in the extraction temperature and extraction power. Figure 3e shows the 3D response surface plots at various extraction temperatures (B) and liquid−solid ratios (D), where the other two parameters are fixed (0 level). The results indicated that the feeding speed of the raw material and the extraction temperature both had a significant impact on the yields. The extraction yields of senkyunolide A, levistolide A, Z-ligustilide, and wallichilide increased sharply with an increase in the extraction temperature (B) and liquid−solid ratio (D). Figure 3f shows the response surface plots at various ultrasound powers (C) and liquid−solid ratios (D) when the remaining parameters are fixed (0 level). The results indicated that the ultrasound power and the liquid−solid ratio both affected the yields, and the liquid−solid ratio had a pronounced impact on the extraction yields. The extraction yields of senkyunolide A, levistolide A, Z-ligustilide, and wallichilide increased sharply with an increase in the ultrasound power (C) and liquid−solid ratio (D). To summarize the results prevented above, the extraction temperature and liquid−solid ratio were found to have a significant influence on the yield, the ultrasound power had an intermediate impact, and extraction time affected the yield to the least extent. Using the optimal balance of factors (extraction time: 30 min, extraction temperature: 45 °C, ultrasound power: 300 W, liquid−solid ratio: 10 mL/g), the total extraction yield obtained from 100 g raw material was 61.12 mg. Separation Results. The CCC and CPC chromatograms obtained using the solvent system comprising n-hexane-ethyl acetate−methanol−water at volume ratios of 4:3:4:2 and 4:3:4:3 are shown in Figure 4. The result indicated that at the volume ratio of 4:3:4:3, the target compounds would have relatively smaller K values and thus would be easily eluted and would not be well-separated. Chromatograms obtained using the solvent system of n-hexane-ethyl acetate−methanol−water
Table 4. Experimental Design Matrix for Screening Variables Affecting the Total Extraction Yield of Senkyunolide A, Levistolide A, Z-Ligustilide, and Wallichilide from Ligusticum chuanxiong Hort, and ANOVA Results sum of squares
source model A, time B, temperature C, power D, liquid− solid ratio AB AC AD BC BD CD A2 B2 C2 D2 residual lack of fit pure error corrected total
degrees of freedom
mean square
F value
p value
20.22 0.57 4.31
14 1 1
1.44 0.57 4.31
26.52 10.46 79.09
