optimization, kinetics, and thermodynamics in the extraction process of puerarin by decompressing...

Optimization, Kinetics, and Thermodynamics in the ExtractionProcess of Puerarin by Decompressing Inner EbullitionXiaoguang Chen,†,‡ Tengyou Wei,†,‡ Mengwei Peng,†,‡ and Zhangfa Tong*,†,‡

†School of Chemistry and Chemical Engineering, Guangxi University, Nanning 530004, China‡Guangxi Key Laboratory of Petrochemical Resource Processing and Process Intensification Technology, Nanning 530004, China

ABSTRACT: Decompressing inner ebullition (DIE) is a novel method and it can achieve a rapid extraction at lowertemperature for active constituents of medical plants. In this work, DIE was employed to extract puerarin from puerarin lobata(Willd.) Ohwi (PLO). The extraction conditions were investigated according single factor analysis. Experiment results showedthat the extraction yield of puerarin reached a maximum value when the ethanol mass concentration in the aqueous solutionimmersing the PLO was 60%, the amount of ethanol solution for premaceration and of water for extraction were 1.6 and 10mL·g−1 of PLO, respectively, the operation gauge pressure was −0.084 MPa, and the extraction temperature was 50 °C. Underthe optimal conditions, the extraction kinetics of DIE was studied. The mass transfer coefficient of DIE ranged between 0.09 and0.13 s−1, which is 183 times greater than that of ultrasonic assistant extraction under the same conditions, and its extractionactivation energy ΔE is 10.86 kJ·mol−1. Also, the thermodynamics properties in the extraction process of DIE at 50 °C were alsocalculated, the enthalpy change ΔH is 58.76 kJ·mol−1, the entropy change ΔS is 224.50 J·mol−1·K−1, and Gibbs free energy ΔG is−13.73 kJ·mol−1, respectively. The extraction of DIE belongs to a spontaneous process with endothermic and entropy increasing.

1. INTRODUCTIONPuerarin lobata (Willd.) Ohwi (PLO) is a famous traditionalmedicinal herb in China, which is also widely accepted byconsumers over the world for its healthy effects. As a principaland major isoflavone, puerarin can be extracted from PLO,1−3

and it has been proven to have notable pharmacologicalfunctions including dilating coronary arteries, decreasingmyocardial oxygen consumption, and improving microcircula-tion in both animals and human patients suffering fromcardiovascular disease.4−6

During the extraction process of puerarin from PLO, theeffect of starch gelatinization must be considered because thatstarch is also a main constituent with 15%∼34.2% content.7

When the temperature is higher than 60 °C, the starchgelatinizes and blocks the catheters within the plant materials,which not only greatly reduces the yield of puerarin, but alsodiminishes the extraction efficiency. To avoid the adverseeffects of starch gelatinization,8,9 conventional methods forextraction of puerarin include organic solvent extraction withaqueous methanol or ethanol using heating reflux or leaching10

techniques for a few to several hours. These procedures haveimportant drawbacks such as long extraction times andconsumption of large quantities of solvents. Recently, severalmodern extraction methods such as microwave assistedextraction (MAE)11,12 and ultrasonic assistant extraction(UAE)13,14 have been used as an alternative to conventionalmethods. MAE and UAE can accelerate the extraction rate andalso can be carried out at lower temperatures below that ofstarch gelatinization. However, these methods require a largeinvestment in equipment and a huge amount of energyconsumption. Thus, they are difficult to be applied for large-scale industry.Decompressing inner ebullition (DIE) is a rapid extraction

method established by Wei et al.15 Generally speaking, DIE

consists of a premaceration and an extraction process underdecompressed pressure. During the premaceration, a smallamount of ethanol solution is used to immerse the raw material,and the inner active constituents in the cells of PLO aredissolved or moistened. Afterward the extraction pressure isdecompressed and the extraction process is strengthened by thevaporization effect of the inner ethanol. The extraction processcan be completed in a short time at lower temperaturesbetween 40 and 60 °C. Good results with respect to extractionefficiency and yield were obtained when the active constituentsof medical plants were extracted by DIE at lower temperaturesbelow that of starch gelatinization.16 Therefore, DIE wasemployed to extract puerarin in this study. After apremaceration with aqueous ethanol, water was used as theextraction solution and the extraction conditions wereoptimized. Moreover, the extraction kinetics and thermody-namics of DIE were investigated. The results provide usefulinformation for extraction of the effective constituents fromherbal medicine with the higher content of starch.

2. EXPERIMENTAL SECTION2.1. Reagents and Materials.17 The roots of PLO were

obtained from Xi’an, China. The reference substance ofpuerarin (95.6%, lot no. 110752-200511) was purchased fromThe National Institute for the Control of Pharmaceutical andBiological Products, Beijing, China. The aqueous ethanolsolutions were prepared by analytical grade ethanol, whichwere used to immerse PLO. Analytical grade methanol wasused to dissolve the extract samples for HPLC analysis, and the

Received: September 5, 2011Revised: April 20, 2012Accepted: April 27, 2012Published: April 27, 2012

Article

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© 2012 American Chemical Society 6841 dx.doi.org/10.1021/ie2020153 | Ind. Eng. Chem. Res. 2012, 51, 6841−6846

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HPLC grade methanol was used as the mobile phase. Both ofthem were purchased from West Chemical, Shantou, China.2.2. DIE. 2.2.1. Experimental Apparatus and Process.18 A

schematic diagram of the DIE extraction apparatus was given inFigure 1. A pressure gauge with a precision of 0.25% MPa and a

vent valve with circular holes were selected to adjust theextraction pressure. A 1 L flask was used as buffer bottle toreduce the deviation caused by the pressure regulator. Theextraction process was as follows: The dried sample powderswere first ground and then sieved through 100-mesh prior touse. The experiments were carried out by placing 10 g ofsample powder in a certain amount of aqueous ethanolsolutions in a 250 mL extraction flask (marked no. 3) andimmersing it for 30 min. Then, some amount of deionizedwater with a desired temperature was added to the no. 3 flask.Sequentially, the pipelines between buffer flask and extractionflask were connected, and the vent valve (marked no. 4 inFigure 1) was used to regulate the extraction pressure.Extracting for a desired time, the extraction pressure wasrelieved and the extraction solution was separated from the

residual of PLO by vacuum filtration. After determining theweight and volume of the extract, 1−1.5 mL samples weretaken by a sampler into a volumetric flask and mixed withchromatographic grade methanol to a metered volume of 25mL. Then, the sample solution was centrifuged for 10 min at10 000 rpm (12 000g). The supernatants were filtered througha 0.45 μm filter membrane and analyzed by HPLC.

2.2.2. Optimization of Extraction Conditions. Severalfactors can impact the yield of puerarin in the process ofextraction by DIE. For increasing the extraction yield ofpuerarin as much as possible, the extraction conditions wereoptimized. The extraction conditions of DIE at different levelsare given in Table 1. According to the single factor analysis, themass concentration of aqueous ethanol solution immersingPLO was first optimized when the amount of ethanol solutionand ratio of water to PLO were set as 1.6 and 10 (v/w), and theextraction temperature and gauge pressure were selected as 50°C and −0.084 MPa, respectively. Similarly, the ratio of ethanolsolution to PLO, the extraction gauge pressure, and the ratio ofwater to PLO were optimized one by one.

2.2.3. Derivation and Determination of Extraction KineticsEquation. During the extraction process by DIE, a largeamount of bubbles generated when the extraction pressure wasdecompressed. It caused the turbulence for particles of PLO inthe solvent. The particles of PLO were contacted ceaselesslywith the solvent, and the puerarin was extracted. This processbelongs to an unsteady-state diffusion process, and it showsgood agreement with the hypothesis of film theory. Therefore,eq 1 was selected, and it was described as follows:19

− = −∞ ∞−q q q b( )/ (1 )e kt

(1)

where t (s) is time of convective mass transfer; q (g) is theamount of the puerarin extracted at time t (then, q = q∞ when t= ∞); b is the washing coefficient, which represents theextraction yield of puerarin recovered instantaneously when

Figure 1. Schematic diagram of the DIE extraction apparatus.

Table 1. Extraction Conditions of DIE at Different Levels by Single Factor Analysis

extraction conditions for optimization levels other conditions for extraction

mass concentrations of aqueous ethanolsolutions for immersing PLO (%)

40 amount of aqueous ethanol solutions immersing PLO, 1.6 mL·g−1; operating gauge pressure, −0.084 MPa; ratioof water to PLO, 10 mL·g−1; extraction temperature, 50 °C50

607080

amount of aqueous ethanol solutionsfor immersing PLO (mL·g−1)

1.2 mass concentrations of aqueous ethanol solutions for immersing PLO, 60%; operating gauge pressure, −0.084MPa; ratio of water to PLO, 10 mL·g−1; extraction temperature, 50 °C1.4

1.61.82.0

operating gauge pressure/MPa −0.086 amount of aqueous ethanol solutions immersing PLO, 1.6 mL·g−1; mass concentrations of aqueous ethanolsolutions for immersing PLO, 60%; ratio of water to PLO, 10 mL·g−1; extraction temperature, 50 °C−0.084

−0.082−0.080−0.078

ratio of water to PLO (mL·g−1) 6 amount of aqueous ethanol solutions immersing PLO, 1.6 mL·g−1; mass concentrations of aqueous ethanolsolutions for immersing PLO, 60%; operating gauge pressure, −0.084 MPa; extraction temperature, 50 °C.8

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Industrial & Engineering Chemistry Research Article

dx.doi.org/10.1021/ie2020153 | Ind. Eng. Chem. Res. 2012, 51, 6841−68466842

PLO is submersed into the solvent and k (s−1) is the masstransfer coefficient.Taking the logarithm of both sides of eq 1 and defining A′ =

ln (1 − b) + ln q∞, eq 2 can be obtained as follows:

− = − + ′∞q q kt Aln( ) (2)

If lnM is subtracted from both sides of the eq 2, whereM (g)is the mass of the extracting solution; eq 2 can be simplified toeq 3:

− = − + ′ −∞q q M kt A Mln[( )/ ] ln (3)

Let C = q/M and A = A′ − ln M, where C (%) is theconcentration of the puerarin in the extraction solution at timet (then, C = C∞ = q∞/M when t = ∞); the extraction kineticsequation is given by

− = − +∞C C kt Aln( ) (4)

To avoid starch gelatinization, three extraction temperaturesfor determining the mass transfer coefficient of DIE werechosen as 40, 50, and 60 °C, respectively. When the extractiontemperature was 50 °C, the extraction kinetic curves of puerarinwere measured under the optimum extraction conditions asfollows: the mass concentration of aqueous ethanol solutionsimmersing the PLO was 60%, the ratios of ethanol solution andwater to PLO were set as 1.6 and 10 (v/w), the operation gaugepressure was −0.084 MPa. Additionally, the extraction kineticcurves of puerarin at 40 and 60 °C under the operating gaugepressure of −0.086 and −0.082 MPa, respectively, were alsodone, and the other extraction conditions were same as that at50 °C.2.2.4. Calculation of Thermodynamics Properties. The

thermodynamic quantities such as change in free energy (ΔG),change in enthalpy of extraction (ΔH), and change in entropy(ΔS) may give an insight into the nature and mechanism ofDIE. The values of ΔH and ΔS were determined from theVan’t Hoff equation as given as follows:20

= − Δ + ΔK H T Sln ( /R ) ( /R) (5)

where R is the gas constant, T is the temperature (in Kelvin),and K is distribution coefficient and is determined as21

= = −K Y Y Y Y Y/ /( )EP RP EP 0 EP (6)

where YEP (%) is the yield of puerarin when the extraction ofDIE achieves equilibrium; YRP (%) is the percent of puerarin inPLO; Y0 (%) is the total content of puerarin in PLO beforeextraction, and it is considered as the sum of puerarin yieldswhen PLO was extracted by DIE for three times.2.3. UAE.22 Ultrasonic power has an important influence on

the extraction rate of UAE. To compare with DIE, theextraction temperature of UAE was selected as 50 °C. It waspresented by Zhou23 that a high yield of puerarin extracted byUAE at 50 °C was obtained when the ultrasonic power was up200 W. To compare with DIE, an experiment was designed andcarried out applying an ultrasonic bath of 40 kHz and 200 W(SK3300LH, kudos Ultrasonic Co. Ltd., Shanghai, China). Theextraction process was as follows: 30.0 g of dried powders and48 mL of 60% aqueous ethanol solutions were added into aflask. (To decrease the effect of sampling, the experimentalscale was enlarged for three times). After 30 min of sampleimmersion, 300 mL of 50 °C water was added, the mixture wastreated with ultrasound simultaneously. At a given duration,0.5∼1.0 mL of extract was removed by a sampler with filter

membrane to a 25 mL volumetric flask. The concentration ofpuerarin within the sample was measured by HPLC, and theextraction kinetic curve was determined. During the entireextraction process, the temperature of water was controlled at50 °C.

2.4. HPLC Analysis and Calculation Method.24 A HPLCanalysis was performed on an Agilent 1200 series HPLC system(Palo Alto, CA, USA) with a E1818009-C18 column (250 mm× 4.6 mm, i.d., 5 μm, Hypersil ODS2). The columntemperature was set at 25 °C, and the analysis was performedusing methanol−water solution (25:75, v/v) as a mobile phase.The flow rate was at 0.8 mL·min−1. The detection wavelengthwas set at 250 nm.The extraction yield of puerarin can be calculated with mass

of the extracted puerarin divided by that of PLO.

3. RESULTS AND DISCUSSION3.1. Optimization of Extraction Conditions. 3.1.1. Con-

centration and Amount of the Aqueous Ethanol SolutionsImmersing PLO. The effects of the concentration and amountof aqueous ethanol solutions immersing PLO were givenrespectively in Figure 2 and Figure 3. It can be seen from Figure

2 that the yield of puerarin has a maximum value with thechange of the mass concentrations of aqueous ethanol solution.

Figure 2. Effect of the mass concentration of aqueous ethanolsolutions immersing PLO on the yield of puerarin by DIE. Otherconditions for extraction were as follows: amount of aqueous ethanolsolutions immersing PLO, 1.6 mL·g−1; operating gauge pressure,−0.084 MPa; ratio of water to PLO, 10 mL·g−1; extractiontemperature, 50 °C (±standard deviations, n = 3).

Figure 3. Effect of the amount of aqueous ethanol solutions immersingPLO on the yield of puerarin by DIE. Other conditions for extractionwere as follows: mass concentrations of aqueous ethanol solutions forimmersing PLO, 60%; operating gauge pressure, −0.084 MPa; ratio ofwater to PLO, 10 mL·g−1; extraction temperature, 50 °C (±standarddeviations, n = 3).



As we know, the polarities of aqueous ethanol solution arerelated to their concentrations. When the polarity of aqueousethanol solution at certain concentration approaches to that ofPLO, the maximum amount of the solvent would permeate intothe cells containing puerarin. Good desorption effects willincrease the transmitted amount of puerarin during thevaporization process of aqueous ethanol solutions as theextraction pressure is decreased. In this work, the optimal massconcentration of aqueous ethanol solutions for immersing PLOwas selected as 60%.The optimal amount of aqueous ethanol solutions used for

immersing PLO is given in Figure 3. A less amount of aqueousethanol solutions cannot maintain a longer time for convectionof DIE, while a larger amount of aqueous ethanol solutions willcause waste. In this process, 1.6 times to the PLO of aqueousethanol solutions was the optimal value.3.1.2. Operation Gauge Pressure and Ratio of Water to

PLO (v/w). To achieve the inner ebullition of the extractionprocess, a proper operation gauge pressure was indispensableunder a certain temperature. Figure 4 is the plot of puerarin

yields under different extraction gauge pressures at 50 °C. Itshows that the yield of puerarin achieved a maximum valuewhen the operating gauge pressure was −0.084 MPa. If theextraction pressure decompresses too little, either the inneraqueous ethanol solutions cannot boil or the intensity ofebullition is insufficient. On the other extreme, if the extractionpressure decompresses too much, the vaporization rate of theinner aqueous ethanol solutions accelerates greatly, whichshortens the duration for DIE. In this case, the extraction yieldof puerarin is reduced. Thus, the optimal operation gaugepressure was selected at −0.084 MPa.The relationship between yields and the ratio of water to

PLO (v/w) is shown in Figure 5. According to the equilibriumprinciple, the more the solvent used, the greater the yield ofpuerarin should be. However, it was shown in experiment thatvolumes of water to PLO up 10:1 (v/w) did not obviouslyaffect the yield of puerarin.3.2. Kinetics of Puerarin Extraction by DIE. The

extraction kinetic curves of DIE were given in Figure 6-I.The extraction kinetic curves of the UAE at 50 °C were shownin Figure 6-II.It can be seen easily from Figure 6, that the extraction

equilibrium of DIE is reached in 40 s, even though theextraction rates differ slightly at different temperatures.

However, 5400 s are needed to reach the equilibriumcomparing with DIE. The total extraction time of DIE is lessthan half time of UAE. Especially for external diffusion rate ofpuerarin in the extraction process, DIE is 135 times faster thanthat of UAE, which is intensified enormously by improvingconvective mass transfer. Also, due to its faster rate of masstransfer, DIE performs with high efficiency over the traditionalmethods. For example, it will take 100 h to leach out thepuerarin completely with the traditional immersing method atroom temperature, and 5 h is needed for the method of heatingreflux with ethanol solvent to extract puerarin.10

Table 2 shows the fitting results of extraction kinetics forboth DIE and the comparison experiment as obtained by eq 4.It can be seen that all fitting coefficients are larger than 0.99.These results indicate that the extraction kinetics of DIE and

Figure 4. Effect of the operating gauge pressure on the yield ofpuerarin by DIE. Other conditions for extraction were as follows:amount of aqueous ethanol solutions immersing PLO, 1.6 mL·g−1;mass concentrations of aqueous ethanol solutions for immersing PLO,60%; ratio of water to PLO, 10 mL·g−1; extraction temperature, 50 °C.(±standard deviations, n = 3).

Figure 5. Effect of the ratio of water to PLO (v/w) on the yield ofpuerarin by DIE. Other conditions for extraction were as follows:amount of aqueous ethanol solutions immersing PLO, 1.6 mL·g−1;mass concentrations of aqueous ethanol solutions for immersing PLO,60%; operating gauge pressure, −0.084 MPa; extraction temperature,50 °C. (±standard deviations, n = 3).

Figure 6. Relationship between extraction time and the concentrationof puerarin in extracts (±standard deviations, n = 3) (I, DIE; II, UAE).

Table 2. Comparison of Parameters and CorrelationCoefficients of Kinetic Equation between DIE and UAE

parameters of the kineticsequations

method temperature (°C) k (s−1) A R2

DIE 40 9.48 × 10−2 −5.65 0.991550 1.08 × 10−1 −5.68 0.992260 1.22 × 10−1 −5.72 0.9918

UAE 50 5.91 × 10−4 −5.72 0.9920



the comparison experiment agree with eq 4, which correspondswith a diffusion model. The mass transfer coefficient k of DIEincreases slightly with increased temperature. The values are allin the range of 0.09∼0.13 s−1. It is affected a little bytemperature, which is due to characteristic of the convectivemass transfer. However, the mass transfer coefficient of UAE is5.91 × 10−4 s−1, which is just 1/183 times to that of DIE at thesame temperature. DIE can accelerate the extraction rate in ashort time mainly owing to the manner change of mass transfer.According to k = k0 exp(−ΔE/RT), the equation ln k = ln k0

− ΔE/RT can be obtained easily. Figure 7 shows the

relationship between ln k and 1/T by DIE. The calculationresult of the active energy is 10.86 kJ·mol−1, which is 34% of theprocess extracted by MAE.25 Therefore, DIE cuts down theenergy barrier in the extraction process and accelerates theextraction rate of puerarin.3.3. Calculation of Thermodynamics Properties for

the Extraction Process. Factually, convection mass transfer isdominant in DIE process, and the extraction process was justaffected a little by temperature. Therefore, eq 5 can be fitted bythe determination of the distribution coefficient at 40, 50, 60°C, and the thermodynamics properties in extraction process ofDIE were calculated. The distribution coefficients at differenttemperatures and the fitting relationship are shown in Table 3and Figure 8.

According to the eq 5, the calculation results of thethermodynamics properties in the extraction process of DIEat 50 °C were shown as follows: ΔH is 58.76 kJ·mol−1, ΔS is224.50 J·mol−1·K−1, and ΔG is −13.73 kJ·mol−1, which wascalculated with the equation of ΔG = ΔH − TΔS.The calculated results indicate that the extraction of DIE

belongs to a spontaneous process with endothermic andentropy increasing.

4. CONCLUSIONSIn this work, a highly effective and green process, DIE, waspresented for extracting puerarin. It achieves a fast extraction of

puerarin at a lower temperature and greatly reduces therequirement of aqueous ethanol solutions. Due to the masstransfer manner of convection, DIE cuts down the energybarrier in the extraction process and enormously accelerates theextraction rate of puerarin. DIE shows a bright prospect forextraction of the effective constituents from herbal medicinewith the higher content of starch.

■ AUTHOR INFORMATIONCorresponding Author*E-mail: [email protected]. Tel.: +86-771-323-9697. Fax: +86-771-323-3718.NotesThe authors declare no competing financial interest.

■ ACKNOWLEDGMENTSPartial support for this work provided by the Scientific andTechnological Project of Guangxi (0718002-4-4) is gratefullyacknowledged. Partial supports for this work provided byScientific and Technological Project Of Guangxi (0718002-4-4)and the Innovation Talent Team Project in GuangxiUniversities (Guijiaoren 2010-38) are gratefully acknowledged.

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Figure 7. Relationship between ln k and 1/T during the extractionprocess of puerarin by DIE.

Table 3. Distribution Coefficients of Puerarin at DifferentTemperatures

40 °C 50 °C 60 °C

ln K 4.43 5.10 5.79

Figure 8. Relationship between ln K and 1/T in the extraction processof DIE.



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