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Separation and Purication Technology 58 (2008) 305310

Pressurized liquid extraction of avonoids from Houttuynia cordata Thunb

Ying Zhang, Shu-fen Li , Xi-wen Wu
Key Laboratory for Green Chemical Technology of State Education Ministry, School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072, China Received 18 August 2006; received in revised form 2 April 2007; accepted 6 April 2007

Abstract The extraction of avonoids from Houttuynia cordata Thunb by pressurized liquid extraction has been studied in this article. The effects of several important factors such as the concentration of slovent ethanol, solvent rate, temperature and pressure were investigated by orthogonal array design experiment. Both a high avonoids yield of 3.152% and a high avoniods content of 23.962% were obtained with solvent ethanol concentration of 50%, solvent rate of 1.8 mL/min, temperature of 70 C and pressure of 8 MPa. The results are favorable to that of traditional methods of hot soaking and ultrasound-assisted extraction. 2007 Elsevier B.V. All rights reserved.
Keywords: Pressurized liquid extraction; Flavonoids; Houttuynia cordata Thunb; Hyperoside; Quercitrin

1. Introduction Houttuynia cordata Thunb is both a medical plant and an edible plant in China. Flavonoids, which display a wide range of pharmacological activities, including antileukemic, antioxidative, antimutagenic, anti-inammatory and antiviral effects as well as the ability of promoting immunologic function, are considered as one kind of the effective components in H. cordata Thunb [14]. And the study of H. cordata Thunb avonoids pharmacological activities also proved them effective components [5,6]. It is of considerable interest to nd a reasonable method to extract and determine the avonoids in H. cordata Thunb. Pressurized liquid extraction (PLE), known as accelerated solvent extraction, which uses a high pressure and temperature, with a forced ow of solvent, is a recently adopted extraction method [7,8]. Compressed nitrogen is usually used as a carrier to move the solvent under high pressure. Increased temperature and elevated pressure accelerate the extraction kinetics so as to enable rapid extraction. Although PLE uses the same aqueous and organic solvents as traditional extraction methods, it makes more efcient use of them.

In the literatures, kinds of traditional methods, such as hot leaching, Soxhlet and ultrasound-asisted extraction have been adopted to extract avonoids from H. cordata Thunb [5,6,914]. But to the knowledge of the author, this is the rst time to study the extraction yield and contents of avonoids in the extracts by PLE. Hot soaking and ultrasound-assisted extraction was also performed as contrast. Single-factor experiment, also called one-variable-at-a-time method, is often used to optimize the experiment conditions. However, it is time-consuming when there are several factors with several levels. Orthogonal array design (OAD) is a kind of statistical based methods, in which orthogonal array is used to assign factors to a series of experimental combinations, whose results can then be treated by range analysis or analysis of variance (ANOVA) [1517]. The use of OAD can simplify the experimental procedure without affecting the quality of the results. 2. Experimental 2.1. Reagents The chemicals of methanol (HPLC grade), acetonitrile (HPLC grade), ethanol, phosphoric acid, sodium nitrite, aluminum nitrate, sodium hydroxide and potassium hydroxide were purchased from Tianjin Keiwei Chemical Factory (Tian-

Corresponding author. Tel.: +86 22 27402720; fax: +86 22 27402720. E-mail address: shi@tju.edu.cn (S.-f. Li).

1383-5866/$ see front matter 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.seppur.2007.04.010

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jin, China). Rutin, hyperoside, quercitrin and quercetin were purchased from the National Institute of the Control of Pharmaceutical and Biological Products, Ministry of Health (Beijing, China). All solutions were ltered through 0.45 m membranes (Chromatography Science and Technology Co., Tianjin, China) before HPLC. Deionized water was used in all experiments. Carbon dioxide (99.95% purity) is used as extraction medium and carrier gas. 2.2. Pretreatment of the raw materials The whole grass of H. cordata Thunb was purchased from Sichuan Province of China, dried to constant weight and pulverized. Before using to extract avonoids, around 900 g of the sample was placed into an extraction vessel with a 5 L capacity. Then supercritical CO2 was used to remove the lipophilic components at 40 C and 20 MPa in a scaling-up SFE system (Huali Pumps Co. Ltd., Hangzhou, China). The remains in the vessel was taken out and stored in airtight black bag as the meterials for this study. 2.3. Extraction methods 2.3.1. Pressurized liquid extraction PLE was performed with Spe-ed SFE system (Applied Separations, Allentown, PA, USA). An accurately weighted portion of around 3 g plant material was mixed with 6 mL corresponding solvent to help it swell and neutral beadings were added

in to play the role of dispersion agent. Then the mixture was placed into a 32 mL stainless steel extraction cell, the two endings of which were padded with several layers of pledget to avoid material being blown out. After the temperature of the cell was approached, the cooled carbon dioxide was delivered passing upwards through the cell at a pressure of around 6 MPa and compressed to the predetermined extraction pressure by an air driven booster pump. Then the sample was subjected to dynamic extraction for 2080 min according to the solvent rate (the volume of solvent is invariably 48 mL each run). The extracts were trapped into a collection vessel and then analyzed by UV for the avonoids yield and its content in the extracts (avonoids content is used for short). It is noteworthy that carbon dioxide was mainly used as carrier to move of the mixture of solvent and solute to the exit for collection. In addition, the extraction medium here, i.e. the mixture of carbon dioxide and aqueous ethanol has higher transfer efciency than aqueous ethanol alone. 2.3.2. Hot soaking To take the literature [18] for reference, accurately weighted portion of around 3 g of plant material, followed by 60 mL of 70% aqueous ethanol was introduced into an airtight Erlenmeyer ask. The asks were then shaken (100 rpm) in a constant temperature water-bath shaker (HZSD, Donglian Electronic Technology Development Co., Haerbin, China) at 70 C for 6 h. Extracts were ltered and then analyzed.

Fig. 1. The molecular structures of quercetin, quercitrin, hyperoside and rutin.

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307

2.3.3. Ultrasound-assisted extraction According to the optimal conditions of the literatures [19], ultrasound-assisted extraction with 70% aqueous ethanol added into around 3 g plant material was performed in an ultrasonic bath with a frequency of 40 kHz (KQ-200DF, Kunshan Ultrasonic Instrument Co. Ltd., China) two times each for 50 min. Extracts were ltered and then analyzed. 2.4. Determination of concentrations of avonoids [14] An aliquot of 6 mL avonoids solution was put into a 25 mL ask, and then 1 mL 5 wt% NaNO2 was added in. After 6 min, 1 mL 10 wt% Al(NO3 )3 was added and after another 6 min, 10 mL 5 wt% KOH was added. After mixing, water was added to the ask to make to the volume. The solution was allowed to stand for 15 min at room temperature, and the absorbance at 500 nm was determined with a UV1600 spectrophotometer (Beijing Ruili Analytical Instrument Co., Beijing, China). Flavonoids concentration was calculated using rutin as the calibration standard. A good linear relationship was obtained over the range of 0.00540.064 mg/mL, and the regression equation is y = 12.80667x 0.00106, where y is the absorbance at 500 nm and x is the concentration of avonoids (mg/mL). 2.5. HPLC of avonoids Before HPLC analysis, all of the samples including the standards were rst dissolved in 70% ethanol. Analysis was performed using a SSI (Scientic Systems Inc., America) HPLC system, consisting of a Model Series III LabAlliance isocratic pump and a Model 500 variable UV/vis detector, linked to a Rheodyne (Rheodyne, Cotati, CA, USA) injector model 7125i equipped with a 20 L sample loop. A Kromasil Turner YWG C18 column (250 mm 4.6 mm i.d., 10 m, B618J0930784) was employed for the separation of samples. Sample injections were effected with a Model 802 RN syringe (10 mL, Hamilton, Bonaduz, Switzerland). The detector was set to 350 nm and the data were recorded and processed with AnaStar chromtography software. H2 OCH3 CNH3 PO4 (400:100:0.2, v/v/v) was adopted as mobile phase in the rst 30 min and then changed to CH3 CNCH3 OHH2 OH3 PO4 (375:75:50:0.1, v/v/v/v) under isocratic conditions. The eluents were ltered through 0.45 m membranes (Chromatography Science and Technology Co., Tianjin, China). Injection volume was 20 L. Flow rate was 1.0 mL/min. All HPLC were performed at 20 1 C. Standard solutions of quercitrin, quercetin, hyperoside and rutin (Fig. 1) were prepared in 70% aqueous ethanol and analyzed by HPLC. The identity of analytes was conrmed by comparing its HPLC retention time with the authentic analytical standard. Quantication was carried out by integration of the peak areas using the external standard method. 3. Results and discussion Various parameters potentially affect the extraction process, so the optimization of the experimental conditions is a critical

Fig. 2. Effect of ethanol concentration, solvent rate, temperature and pressure on avonoids yield.

step in the development of a PLE method to extract avonoids from H. cordata Thunb. Orthogonal experiments were carried out to examine the factors, such as concentration of ethanol, solvent rate, temperature and pressure, the selection of which was based on the previous knowledge about PLE [20,21]. As is known, compared with methanol, chloroform, ethanol is a less-toxicity and acceptable for processing medicines. By regulating the ration of water and ethanol, the properties of the uids can be readily manipulated. Therefore, aqueous ethanol was chosen as the solvent. A four-level OAD with an OA16 (45 ) matrix was chosen. Table 1 shows the OAD factors and its levels for the extraction of avonoids. Here the interactions among different variables were neglected and focus was placed on the main effects of the four most important factors. The results, which are the average values of two runs, are shown in Table 2. 3.1. Experimental designed data analysis The results shown in Table 2 indicate that there are great differences of yield or content among each set of PLE conditions. If the avnoids yield and content were expressed as control indexes, the mean values of the yield or content for the factors at each level were calculated and the results are shown in Figs. 2 and 3. For example, the mean value of the yield for the temperature at level 1 is calculated as (0.548 + 1.388 + 1.982 + 1.467)/4. Variance analysis on the data from the orthogonal experiments was applied in Table 3 [22]. From Table 3, it is seen that avonoids yield and content being the indexes, ethanol concentration is statistically the most signicant at P < 0.01. Solvent rate is signicant to yield at P < 0.25 and temperature is signicant to content at P < 0.10. As the results of varience analysis shown, neither yield nor content is signicantly affected by pressure, so a lower pressure of 8 MPa was adopted in the following experiments.

308 Table 1 OAD factors and its levels Levels Factors

Y. Zhang et al. / Separation and Purication Technology 58 (2008) 305310

A: Ethanol concentration (%) 1 2 3 4 0 30 50 70

B: Solvent rate (mL/min) 0.6 1.2 1.8 2.4

C: Temperature ( C) 40 50 60 70

D: Pressure (MPa) 8 12 16 20

Table 2 Results of OA16 (44 ) Test number Factors A: Ethanol concentration (%) 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
a b

Yielda (%) B: Solvent rate (mL/min) B1 B2 B3 B4 B1 B2 B3 B4 B1 B2 B3 B4 B1 B2 B3 B4 C: Temperature ( C) C1 C2 C3 C4 C2 C1 C4 C3 C3 C4 C1 C2 C4 C3 C2 C1 D: Pressure (MPa) D1 D2 D3 D4 D3 D4 D1 D2 D4 D3 D2 D1 D2 D1 D4 D3 0.548 0.572 0.828 0.944 1.311 1.388 2.117 1.867 2.191 2.052 1.982 2.086 1.183 1.569 1.798 1.476

Contentb (%)

A1 A1 A1 A1 A2 A2 A2 A2 A3 A3 A3 A3 A4 A4 A4 A4

5.164 5.129 6.338 8.226 13.946 14.350 16.117 15.073 17.514 18.505 15.955 16.359 18.495 13.595 15.061 12.832

Flavoniods yield (%) = (the amount of avonoids/material mass) 100. Flavonoids content (%) = (the amount of TF/the amount of crude extracts) 100.

3.2. Effect of ethanol concentration One of the key steps of solvent extraction process is that the target compounds dissolve in the chosen solvent. According to the principle of like dissolves like, solvent of a polarity which is similar to that of the target compounds is likely to dissolve more of the latter. PLE was performed using ethanol of different concentrations in water (0, 30, 50, 70%) as solvent. These different concentrations led to different polarities of the solvent. Figs. 2 and 3 demonstrate that an ethanol concentration of 50% results in both a highest avonoids yield and a highest

avonoids content. The widest ranges of the broken line plots of ethanol concentration in the direction of Y-axis also indicate the signicance of this factor. 3.3. Effect of solvent rate Since the volume of the solvent was invariable in these experiments, an increase of solvent rate meant a decrease of the dynamic extraction time. The effect of solvent rate on the extraction results is shown in Figs. 2 and 3. As shown in Fig. 2, when the solvent rate is increased, the balance of

Table 3 ANOVA table for the PLE of avonoids from Houttuynia cordata Thunb by using OA16 (44 ) matrix Source of variance Sum of squares Yield Ethanol concentration Solvent rate Temperature Pressure Pooled error Total
a b c

Degree of freedom Content 279.463 1.721 24.521 3.066 2.653 311.424 3 3 3 3 3 15

Mean square Yield 1.289 0.119 0.060 0.039 0.041 Content 93.154 0.574 8.174 1.022 0.884

F-value Yield 31.063a 2.865b 1.435 0.946 Content 105.321a 0.648 9.241c 1.156

3.866 0.357 0.179 0.118 0.124 4.644

P = 0.01 (99% condence level); F(3,3) = 29.46. P = 0.25 (75% condence level); F(3,3) = 2.36. P = 0.10 (90% condence level); F(3,3) = 5.39.

Y. Zhang et al. / Separation and Purication Technology 58 (2008) 305310 Table 4 Results contrast of 70 and 80 C (n = 2) Temperature ( C) 70 80 Yield (%) 3.152 3.277

309

Content (%) 23.962 22.909

the optimal conditions (50% ethanol, solvent rate 1.8 mL/min, pressure 8 MPa), and shows that the extracts content of 80 C is not higher than that of 70 C, which may be attributed to that more other components were extracted at a higher temperature. So 70 C is the reasonable temperature. 3.5. Comparison of PLE with hot soaking and ultrasound-assisted extraction
Fig. 3. Effect of ethanol concentration, solvent rate, temperature and pressure on avonoids content.

extraction shifted in the favor of improving avonoids yield. However, a further increase in the solvent rate resulted in decreased extraction efciency. This is due to the short extraction time corresponded to the high solvent rate, which leads to insufcient mass transfer between the solid material and the solvent. Fig. 2 indicates 1.8 mL/min is the most suitable solvent rate. 3.4. Further experiment of temperature In the range of the experiment, the content increases as the temperature increases (as shown in Fig. 3), so the optimization experiment of temperature was extended to a higher temperature, i.e. 80 C. Table 4 contrasts the results of 70 and 80 C under

Hot soaking and ultrasound-assisted extraction were performed as what referred in Section 2. The results are exhibited in Table 5, which also gives the HPLC determination results of rutin, hyperoside, quercitrin and quercetin. A typical HPLC chromatogram of sample extracted by PLE is displayed in Fig. 4. The chromatograms of samples extracted by the other two methods are similar in the shape and location of the peaks but different in the peak areas. The results in Table 5 demonstrate the obviously shorter extraction time, higher avonoids yield and content of PLE. The HPLC results show that each method is able to extract these identied avonoids. Though the yield of rutin is a little lower compared with the other two methods, PLE has an obvious advantage in the yield of hyperoside, quercitrin and quercetin, whose contents in H. cordata Thunb is much higher than rutin. Moreover, compared with the batch wise operation of hot soaking and ultrasound-assisted extraction, the semi-batch wise operation of PLE is also strength of this method.

Fig. 4. HPLC chromatograms of sample extracted by PLE. 1, Rutin; 2, hyperin; 3, quercitrin; 4, quercetin.

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Table 5 Yield and content of avonoids extracted by different methods (n = 2) Methods Extraction time (min) Yielda (%) Content (%) Yielda (mg/g) Rutin Hot soaking Ultrasound-assisted extraction PLEb
a b

Hyperoside 0.996 1.040 1.528

Quercitrin 1.497 1.653 1.724

Quercetin 0.314 0.279 0.524

360 100 27

1.925 1.929 3.152

19.333 15.374 23.962

0.391 0.355 0.336

Extraction yield (mg/g) = the amount of target compounds/material mass. PLE results is the results of the optimal conditions. [6] J.Y. Qiu, Y.L. Yang, G.R. Yang, Q. Lin, Q. Zhou, J. Yunnan Univ. 27 (3) (2005) 239244. [7] B.E. Richter, J.L. Ezzell, D.E. Knowles, F. Hoeer, A.K.R. Mattulat, M. Scheutwinkel, Chemosphere 34 (1997) 975987. [8] B.E. Richter, J. Chromatogr. A 874 (2000) 217224. [9] J.X. Zhang, Anhui Agric. Sci. Bull. 6 (11) (2005) 5152. [10] L.K. Su, Y.J. Hu, L.C. Fu, Y.T. Zhu, Tradit. Chin. Drug Res. Clin. Pharmacol. 10 (4) (1999) 232234. [11] J.M. You, D.Y. Liu, Food Res. Dev. 25 (6) (2004) 5556. [12] X.M. Li, W.L. Xu, X.R. He, J. Guangdong College Pharm. 21 (2) (2005) 140142. [13] P. Tang, Y. Li, Jiangsu J. Tradit. Chin. Med. 26 (4) (2005) 40. [14] Q. Sheng, Y.C. Ren, Y.G. Zhou, J. Zhejiang Med. Acad. Sci. 40 (1999) 1920. [15] G. Zhu, H. Ju, Anal. Chim. Acta 506 (2004) 177181. [16] N. Bahramifar, Y. Yamini, M. Shamsipur, J. Supercrit. Fluids 35 (2005) 205211. [17] W. Gu, C.Y. Zhou, M.K. Wong, L.M. Gan, Talanta 46 (1998) 10191029. [18] J. Cai, J.Q. Hua, W. Wang, J. Huaiyin Inst. Technol. 12 (5) (2003) 82. [19] X.G. Shi, X. Wang, Y.L. Geng, J. Shandong Inst. Light Ind. 18 (2) (2004) 56. [20] M. Christophe, L. Ga tane, P.G. Martine, Anal. Bioanal. Chem. 382 (2005) e 15741583. [21] M. Waksmundzka-Hajnos, A. Petruzynik, A. Dragan, D. Wianowska, A.L. Dawidowicz, Phytochem. Anal. 15 (2004) 313319. [22] Y.Y. Feng, Basic Experiments of Chemical Engineering, rst ed., Chemical Industry Press, Beijing, 2000.

4. Conclusion Optimal conditions of PLE of H. cordata Thunb avonoids, i.e. solvent of 50% ethanol, solvent rate of 1.8 mL/min, a temperature of 70 C and a pressure of 8 MPa, were found through an orthogonal test and a single-factor experiment. Under these optimal conditions, the avonoids yield and content can reach 3.152 and 23.962%, respectively. A comparison between PLE, hot soaking and ultrasound-assisted extraction demonstrated the higher extraction yield and extracts content of PLE. It can be concluded that PLE is very useful technique for the extraction and isolation of avonoids from H. cordata Thunb. References
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