Comparison of modified ultrasound-assisted and traditional extraction methods for the extraction of baicalin and baicalein from Radix Scutellariae

Industrial Crops and Products 45 (2013) 182–190

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Comparison of modified ultrasound-assisted and traditional extraction methods for the extraction of baicalin and baicalein from Radix Scutellariae Yu-Chiao Yang a , Ming-Chi Wei b,∗ , Ting-Chia Huang c , Suen-Zone Lee b , Shiow-Shyung Lin b a b c

Department and Graduate Institute of Pharmacology, Kaohsiung Medical University, Kaohsiung 80708, Taiwan Department of Environmental Engineering & Science, Chia Nan University of Pharmacy and Science, Tainan 71710, Taiwan Department of Chemical Engineering, National Cheng Kung University, Tainan 70101, Taiwan

a r t i c l e

i n f o

Article history: Received 8 August 2012 Received in revised form 25 November 2012 Accepted 30 November 2012 Keywords: Radix Scutellariae Ultrasound-assisted extraction HPLC Baicalin Baicalein

a b s t r a c t An attempt was made in this study to extract baicalin and baicalein from Radix Scutellariae using modified ultrasound-assisted extraction (UAE), and these results were compared to a commonly used heat reflux extraction (HRE) procedure. The best results were obtained when working at an ultrasonic frequency of 40 kHz, a power of 185 W, an ultrasound cycle of 79% (intermittent sonication), a mean particle size of 0.355 mm, a ratio of solvent to raw material of 10:1 (mL/g), an extraction time of 20 min and three cycles. The optimum extraction temperature and ethanol concentration differ for different flavanone compounds. The extraction of baicalin and baicalein were most efficient at temperatures of 60 and 30 ◦ C, respectively, with ethanol concentrations of 40 and 50% (v/v), respectively. Compared with HRE, the modified UAE method reduced the extraction time, the extraction temperature and the solvent consumption. Moreover, UAE achieved superior baicalin and baicalein yields, is a potential method for industry. © 2012 Elsevier B.V. All rights reserved.

1. Introduction Radix Scutellariae (Huang-Qin in China), an important traditional Chinese medicine, is derived from the dried root of a medicinal plant named Scutellaria baicalensis Georgi (Labiatae). In traditional medicine, it is extensively used in treatments for inflammation, cancer, hepatitis, cirrhosis, diarrhea, jaundice, painful micturition, hematemesis, epistaxis, hematuria, metrorrhagia, cardiovascular dysfunction, infections of the respiratory and the gastrointestinal tract, cleaning away heat, moistening aridity, purging fire, detoxifying toxicosis, reducing the total cholesterol level and decreasing blood pressure (Li et al., 2004; Wang et al., 2010). Recent studies have demonstrated that Radix Scutellariae reveals a good antitumor effect, which can be used for the treatment of human breast cancer (Tayarani-Najaran et al., 2010; Wang et al., 2010), prostate cancer (Lee et al., 2008), leukemia (Özmen et al., 2010), hepatocellular carcinoma (Wang et al., 2006), cervical carcinoma (Tayarani-Najaran et al., 2010), lung carcinoma (Du et al., 2010), colorectal carcinoma (Lee et al., 2008), bladder cancer (Ikemoto et al., 2000), lymphoma (Kumagai et al., 2007), myeloma (Kumagai et al., 2007) and gastric adenocarcinoma (Tayarani-Najaran et al., 2010; Zhao et al., 2010). Further reports have shown that the extract generally contains a large amount of flavonoids such as baicalin and

∗ Corresponding author. Tel.: +886 6 2664911x6215; fax: +886 6 2667320. E-mail address: [email protected] (M.-C. Wei). 0926-6690/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.indcrop.2012.11.041

baicalein (Kido et al., 2008). Flavonoids have received much attention for their curative effects, including their anti-tumor (Middleton et al., 2000; Chou et al., 2009; Li-Weber, 2009; Özmen et al., 2010), hepatoprotective (Sun et al., 2010), anti-bacterial (Sato et al., 2000), anti-inflammatory (Cui et al., 2010) and antioxidant activities (Yao et al., 2009), as well as for their abilities to reduce the risk of cardiovascular diseases (Middleton et al., 2000; Lee et al., 2011). Among these compounds, baicalin (Li-Weber, 2009; Du et al., 2010; Xu et al., 2011) and baicalein (Chou et al., 2009; Li-Weber, 2009; Özmen et al., 2010; Wang et al., 2010) have a broad range of biological effects with a low toxicity and are among the most promising chemopreventive agents for cancer. These findings have made it an attractive dietary supplement in an expanding health food market since this two compounds have already occurred widely in food that consumed by humans. Therefore, it is interesting to find an effective method to extract baicalin and baicalein from Radix Scutellariae (Fig. 1). Due to the complex composition of a raw herbal extracts and the presence of interfering compounds, various analytical methods have been reported for the quantification of bioactive constituents in herbal plants. The qualitative and quantitative analysis methods of flavonoids in various matrices are high performance liquid chromatography (HPLC) (Ohkoshi et al., 2009; Dykes et al., 2011), liquid chromatography–electrospray ionization mass spectrometry (LC–ESI–MS) (Piccinelli et al., 2011), and liquid chromatography–mass spectrometry (LC–MS) (Biesaga, 2011). To evaluate the quality control of Radix Scutellariae, the development

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The objective of this study was to obtain UAE extracts from Radix Scutellariae and to discover the best conditions for extracting baicalin and baicalein. To achieve these goals, we studied the influence of the process parameters, namely, the duty cycle of ultrasound exposure, particle size, extraction time, extraction temperature, ratio of solvent to raw material, ethanol concentration and number of extraction cycles, on the extraction yields of the target compounds. Analytical determinations of both compounds were carried out by HPLC. The conventional heat reflux of baicalin and baicalein from Radix Scutellariae was also carried out and treated as a control. 2. Materials and methods 2.1. Chemicals and reagents

Fig. 1. Structures of baicalin and baicalein.

of an HPLC analytical method to simultaneously determine target compounds in the herbal extract is preferred. Several reports in the primary literature have discussed the analysis of flavonoids in other herbal medicines using HPLC methods (Ohkoshi et al., 2009; Dykes et al., 2011). However, information on the use of HPLC for the identity and quantity of baicalin and baicalein from Radix Scutellariae is rarely reported. It is well known that the pharmacologically active compounds in herbal plants are usually present in low concentrations and are composed of complex chemical constituents. For the recovery and purification of desired compounds from raw matrices, selecting the appropriate extraction method is a key consideration. Conventional techniques such as heat reflux and Soxhlet extraction can be used to extract natural flavonoids from various matrices. However, there are disadvantages, including the consumption of large volumes of solvent and energy, lengthy extraction times, and the potentially deleterious degradation of labile compounds. At present, proposed extraction methods capable of overcoming the above-mentioned drawbacks include UAE (Briars and Paniwnyk, 2013; Khan et al., 2010; Yang et al., 2012), microwave-assisted extraction (Liao et al., 2008), and supercritical fluid extraction (Lee et al., 2010). Among these methods, UAE has been widely employed in the extraction of target compounds from different materials, owing to its facilitated mass transport of solvent from the continuous phase into plant cells (Vinatoru, 2001). In addition, the energy consumption of UAE could be effectively decreased by employing a lower processing temperature and shorter extraction times. In theory, swelling and hydration can be accelerated by ultrasound, thus resulting in a probable enlargement in the pores of cell walls and allowing target compounds to be more easily released from the matrix into the extraction medium (Sun and Tomkinson, 2002). Therefore, UAE allowed target components to dissolve in the extraction medium at higher rates, thereby boosting yield in comparatively less time. Furthermore, in comparison with other extraction techniques such as microwave-assisted extraction and supercritical fluid extraction, the equipment of UAE is simpler and economically cheaper. The UAE process is widely used as an attractive alternative extraction method to conventional liquid extraction in wide variety areas including the industries of food, pharmacy and environmental engineering. However, there have not been reports on the application of UAE for the extraction of baicalin and baicalein from Radix Scutellariae.

The flavone standards, baicalin (98%) and baicalein (98%), were purchased from Aldrich Chemical Co. (Milwaukee, WI, USA). Methanol (99.9%), ethanol (99.9%), acetone (99.7%), acetonitrile (99.9%), ethyl acetate (99.9%), n-hexane (95%) and 85% phosphoric acid were bought from Merck Co. (Darmstadt, Germany). Deionized water was prepared using a Milli-Q reverse osmosis unit from Millipore (Bedford, MA, USA). 2.2. Plant material Three batches of dried whole-plant materials from Radix Scutellariae (named sample RS1, RS2 and RS3) were generously provided by Kaiser Pharmaceutical Limited Company (KPC, Tainan, Taiwan). All samples were sorted and identified by the Research and Development Division of KPC, and the quality complied with that of the Chinese Pharmacopoeia. All voucher specimens (RS1 to RS3) have been deposited at the Laboratory of Department and Graduate Institute of Pharmacology, Kaohsiung Medical University (Kaohsiung, Taiwan). The air-dried whole plants were pulverized in a knife mill, and parts of the plants were sieved to different sizes of 0.925, 0.725, 0.550, 0.355 and ≤0.21 mm (mean diameter) and subsequently packed into plastic bags and stored at 4 ◦ C in a refrigerator for later use. In addition, the moisture content (% dry weight basis) was determined by drying at 105 ◦ C to a constant mass and was 12.45%. All yields and composition analyses were calculated based on a moisture-free basis and represent the mean values of at least six experiments. 2.3. Modified ultrasound-assisted extraction (UAE) UAE was carried out using an ultrasonic cleaning bath with a working frequency of 40 kHz and 185 W of power (Branson B-33510E-DTH, USA); this extraction was operated in a pulse mode (intermittent sonication) that provided 95 s of pulse on followed by 25 s of pulse off for every 120 s (79% on/off time, work with the hands). Briefly, the bath was a rectangular container (290 mm × 240 mm × 150 mm) connected to a temperature-controlled water bath (Haake F3-K, Haake, Karlsruhe, Germany). Five grams of dried plant powder were loaded in a 250 mL flask (flat bottom) containing a volume of extraction solvent according to the experimental design. The flask was then immersed into the ultrasonic bath (fixed at same position of the ultrasonic bath) to maintain a constant temperature throughout the period of extraction. The flask located at the point where the maximum sonochemical effect is achieved (Mason, 2000). To prevent the loss of solvent during extraction, a condenser was connected to the flask. The water in the ultrasonic cleaner bath was circulated and regulated at a constant temperature to prevent any rise in the water temperature caused by exposure to ultrasonic waves. Aqueous solutions with various concentrations of ethanol were used as

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the extracting solvent, and the extraction was performed according to the experimental design under various UAE conditions. The choice of an aqueous ethanol solution as the extracting solvent was based on its polarity relative to the target compounds and its acceptability for practical use in the food, cosmetic and pharmaceutical industries. The temperature of the flask was measured with a mercuryin-glass thermometer having ±0.1 ◦ C divisions. The extraction experiments were started when the system reached the desired temperature. There were two stages of extraction: heat reflux extraction (5 min) and ultrasound-assisted extraction (5–60 min). A heat reflux extraction with an immersion-stirring device (200 rpm; Thermo Scientific Variomag Compact and Maxi, USA) was used to obtain good solvent-to-plant material contact. After heat reflux extraction, the immersion-stirring device was drawn from ultrasonic cleaning bath. The UAE conditions were then performed at various ratios of solvent to raw material (6–22 mL/g), ultrasound cycle (0–100%), ethanol concentrations (0–100%), mean particle sizes (0.925, 0.725, 0.550, 0.355 and ≤0.21 mm), extraction times (5–60 min), extraction temperatures (30–80 ◦ C) and extraction cycles (1–4). After extraction, the solutions were carefully decanted, and the plant material was re-extracted with the same solvent. The extracts from each set were combined and vacuumfiltered while hot using Whatman No. 1 filter paper. The filtrate was dissolved in 5 mL of methanol and was then concentrated in a rotary evaporator (Eyela Rotary Evaporator N-1000, Tokyo Rikakikai Co., Ltd, Japan) under reduced pressure. The resulting solution was filtered through a 0.45-␮m syringe filter (Millipore, Billerica, MA, USA) before HPLC analysis. At least six replicates were performed for each extraction condition tested, and all analyses were repeated six times. 2.4. Heat reflux extraction (HRE) HRE was performed by mixing 5 g of dried, powdered plant with an appropriate amount of extraction solvent in a 250 mL flask (flat bottom), which was immersed in a temperature-controlled water bath while stirring (200 rpm). The extraction temperature (30–80 ◦ C), duration (10–120 min), ratio of solvent to raw material (6–22 mL/g), ethanol concentration (0–100%, v/v), mean particle sizes (0.925, 0.725, 0.550, 0.355 and ≤0.21 mm) and extraction cycles (1–4) were optimized. 2.5. High-performance liquid chromatography (HPLC) HPLC analysis of target compounds was conducted on a Jasco HPLC system (Jasco, Tokyo, Japan) with a LiChrospher® C18 analytical column (250 mm × 4 mm i.d., 5-␮m particle size; Merck, Darmstadt, Germany). The mobile phase was composed of acetonitrile (solvent A) and 0.1% phosphoric acid (solvent B), and the following gradient was used: 0–8 min, 22% (solvent A); 8–25 min, 22–23% (solvent A); 25–50 min, 23–43% (solvent A). Also, the following flow rate was used: 0–25 min, 1.0–1.5 mL/min; 25–32 min, 1.5–1.0 mL/min; 32–50 min, 1.0 mL/min. The column was equilibrated for 20 min prior to each analysis. A 20-␮L sample and a standard solution were individually injected into the chromatographic system. The temperature of the column was maintained at 40 ◦ C, and the eluent was monitored at 280 nm. The quantities of the target compounds were calculated by comparing their peak areas to those of the standards. 2.6. Recovery To determine the extraction recoveries of baicalin and baicalein from Radix Scutellariae, the contents of the two analytes in a sample were estimated according to their respective calibration curve

Fig. 2. The HPLC chromatograms of (A) the reference compounds and (B) a Radix Scutellariae extract obtained by UAE.

(baicalein: Y = 15421.52 X – 669502.26, R2 = 0.999, 50–200 ␮g/mL; baicalin: Y = 7589.906 X – 24084.944, R2 = 0.999, 10–180 ␮g/mL). Three different quantities (low, medium, and high) of the authentic standards were added to the sample solution before extraction. The follow-up extractions and HPLC analyses were performed in the same manner as above. At least six replicates were performed for each extraction condition tested, and all analyses were repeated six times. The recovery for each analyte was evaluated by comparing the difference of the analyte mass between the fortified samples and the original sample with the nominal mass of the analyte spiked in the fortified samples. The recovery was calculated using the following formula: Recovery (%) = [(found value–content)/spiked value] × 100. 2.7. Statistical analysis All yields and composition analyses were calculated based on a moisture-free basis. The mean and standard deviation (SD) of the mean were calculated from six experiments. The results are expressed as the mean ± standard SD. Analysis of variance (ANOVA) was carried out using Tukey’s method with a significance level of p < 0.05 using 2010 Microsoft Office Excel (Microsoft CO., USA) and Origin version 6.1 software (Origin Lab CO., Northampton, MA, USA)

3. Results and discussion This work was designed to develop a flavonoid extraction method, as well as a method based on reverse phase HPLC separation combined with UV detection for a flavonoid assay in Radix Scutellariae extracts. The analytical method described above was used to analyze the target compounds. Baicalin and baicalein standards were detectable under a wavelength of 280 nm. At this wavelength, baicalin and baicalein could be eluted efficiently and simultaneously detected by a mobile phase consisting of acetonitrile (solvent A) and 0.1% phosphoric acid (solvent B). The chromatographic retention times of baicalin and baicalein were 16.64 ± 0.027 and 43.65 ± 0.075 min, respectively (Fig. 2A). This HPLC system successfully separated and simultaneously identified baicalin and baicalein in the Radix Scutellariae extract (Fig. 2B), and it can be used to quantify the content of baicalin and baicalein.

Y.-C. Yang et al. / Industrial Crops and Products 45 (2013) 182–190 Table 1 The extraction yields of the target compounds obtained using the HRE and conventional UAE methods with various solvents. Extraction method/Solvent

Extraction yield (mg/g)a Baicalin

Baicalein

HHRE Water 5% Ethanol 10% Ethanol 20% Ethanol 30% Ethanol 40% Ethanol 50% Ethanol 60% Ethanol 70% Ethanol 80% Ethanol 90% Ethanol 95% Ethanol 10% Ethyl acetate 20% Ethyl acetate 30% Ethyl acetate 40% Ethyl acetate 50% Ethyl acetate 60% Ethyl acetate 70% Ethyl acetate 80% Ethyl acetate 90% Ethyl acetate 99.9% Ethyl acetate

Not detected Not detected 2.6766 ± 0.1285 43.4944 ± 1.6484 49.5019 ± 1.9850 54.4238 ± 1.8994 54.8699 ± 2.0576 50.3941 ± 1.8847 47.7323 ± 1.7375 28.1041 ± 1.1017 14.7212 ± 0.5476 8.0297 ± 0.2818 47.8723 ± 1.7569 45.7447 ± 1.8023 44.6809 ± 1.4253 40.4255 ± 2.1938 36.3830 ± 1.4881 28.7234 ± 1.3213 2.1277 ± 0.0981 1.4894 ± 0.0541 1.2766 ± 0.0527 1.0302 ± 0.0385

Not detected Not detected 2.2305 ± 0.1149 6.2454 ± 0.2436 9.8141 ± 0.4239 12.0446 ± 0.4842 11.1524 ± 0.4361 12.4907 ± 0.4821 13.3829 ± 0.5447 15.1673 ± 0.6552 16.5056 ± 0.7361 14.2751 ± 0.5967 9.3617 ± 0.4693 9.5745 ± 0.3150 11.2766 ± 0.3913 12.7660 ± 0.4838 13.6170 ± 0.5610 19.5745 ± 0.8084 26.2979 ± 0.8389 26.9362 ± 1.1144 27.9028 ± 1.3421 9.9382 ± 0.4095

Conventional UAEc 60% Ethanol

45.7074 ± 1.8329

11.3291 ± 0.4430

b

a

The extraction yields of the target compounds are expressed as mg/g of plant dry weight basis. Values are given as the mean ± SD (n = 6). b Experimental conditions of HRE method: extraction time: 60 min; temperature: 60 ◦ C; solvent to raw material: 14:1 mL/g; mean particle size: 0.355 mm; stirring: 200 rpm. c Experimental conditions of conventional UAE method: extraction time: 60 min; temperature: 60 ◦ C; solvent to raw material: 12:1 mL/g; mean particle size: 0.355 mm; stirring: 0 rpm.

3.1. Heat reflux extraction For the preparation of crude extracts from plants, a selection of the appropriate extraction solvent is a key consideration. For this purpose, a method based on heat reflux leaching with subsequent HPLC analysis was firstly investigated and optimized. The effects of several solvents, including ethyl acetate, aqueous ethyl acetate (10–90%), ethanol, aqueous ethanol (5–90%) and water, on the extraction efficiencies of baicalin and baicalein from Radix Scutellariae were evaluated as shown in Table 1 (one extraction cycle). The extraction yields of baicalin and baicalein are expressed as mg/g Radix Scutellariae. HRE were performed at a temperature of 60 ◦ C with a mean particle size of 0.355 mm, a ratio of solvent to raw material of 14:1 mL/g and stirring at 200 rpm for 60 min. It is evident from Table 1 that the yields of baicalin and baicalein were dependent on different solvents. The choice of solvents used was made based on their polarity relative to baicalin and baicalein. Because baicalin and baicalein are polar compounds, polar solvents were selected. Of all the solvents tested, ethanol and water are acceptable for practical use, as they were in compliance with good manufacturing practices. As revealed by the results, the optimum extraction solvent for each flavonoid was different. For example, according to Table 1, the most effective extraction solvent for baicalin was 50% ethanol, whereas 90% ethyl acetate extracted the highest yield of baicalein. This difference could be due to differing polarities and viscosities. Moreover, the aqueous solution system clearly had a much better extraction performance than any of the other solvents. The high performance of the aqueous systems may be ascribed to the solvent affinity between flavonoids and the mixed solvent system of ethanol (or ethyl acetate) and water being higher than the individual affinity of each. The results may be attributed to

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solvent–solute affinity and the effective swelling of the plant material by the solvent, which helped increase the surface area for solute–solvent contact. Furthermore, the presence of water lowers the viscosity of the mixed solvent system, and thus, mass transfer was improved. However, ethyl acetate is a toxic solvent. We therefore selected aqueous ethanol (0–100%) as the extraction solvent for subsequent investigations. 3.2. Optimization of ultrasound-assisted extraction UAE is a relatively inexpensive technique and has lower instrumental requirements. The advantages of UAE are significantly reduced extraction times, decreased extraction temperature and increased extraction yields. Therefore, UAE is offered as an alternative process to HRE for obtaining baicalin and baicalein from Radix Scutellariae in this study. However, the experimental results (shown in Table 1) were still not satisfied the extraction yields of baicalin and baicalein in the extracts compared to that of HRE. Therefore, modified ultrasound-assisted extraction in the presence of two stages of extraction was employed in order to enhance the extraction efficiency. Because various parameters potentially affect the UAE process, establishing appropriate extraction parameters relating to specific plant materials is very important. Moreover, optimizing these conditions to increase efficiency and to lower solvent and energy consumption has economic benefit. From previous reports (Cuoco et al., 2009), particle size was found to affect the extraction efficiency of target compounds from raw materials. In our previous study (Yang et al., 2012), particle size was found to affect the extraction efficiencies of oridonin, oleanolic acid and ursolic acid from Rabdosia rubescens. The extraction yield increased as the mean particle size decreased from 0.925 to 0.355 mm and slightly decreased as the mean particle size decreased from 0.355 to ≤0.21 mm under UAE. The slightly lower yield observed with particles of smaller size (≤0.21 mm) could be due to the particles staying at the surface of the solvent during extraction, resulting in oridonin, oleanolic acid and ursolic acid being less easily released from Rabdosia rubescens into the extraction medium. Therefore, a mean particle size of 0.355 mm was selected as optimum for this study. To achieve the best extraction efficiencies of baicalin and baicalein from Radix Scutellariae, the extraction conditions, including extraction times, extraction temperatures, ultrasound cycle, liquid/solid ratio, mean material particle size and compositions of ethanol in ethanol–water mixtures, were further studied and optimized. The experiments were performed by varying the experimental parameters one at a time while all other variables remained fixed. Optimal conditions were identified as those that afforded the highest extraction yields of the target compounds. 3.2.1. Effect of extraction temperature First, to determine the effect of the extraction temperature on extraction yields, extraction experiments were conducted at temperatures ranging between 30 and 80 ◦ C. The UAE experiments were set as follows: the ratio of solvent to raw material was 10:1 (mL/g), the ultrasound cycle was 79%, the mean material particle size was 0.355 mm, the extraction time was 20 min, and the ethanol concentration was 40% for baicalin and 50% for baicalein. The effects of the extraction temperature on the extraction yields of baicalin and baicalein from Radix Scutellariae are shown in Fig. 3 (one extraction cycle). As shown in Fig. 3, the most efficient extraction temperature was different for baicalin and baicalein. The results indicated that the maximum extraction yield for baicalin was at 60 ◦ C for 20 min and that for baicalein was at 30 ◦ C for 20 min. These results are consistent with those reported by other authors using different plant materials (Satsuma Mandarin Peels) by ultrasonic treatment, varied from one compound to another (Ma et al., 2008a).

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Fig. 3. Effect of extraction temperature on the extraction yields of baicalin and baicalein from Radix Scutellariae (one extraction cycle). Extraction time: 20 min; ultrasound cycle: 79%; ethanol concentration: 40% (v/v) for baicalin, 50% (v/v) for baicalein; mean particle size: 0.355 mm; ratio of solvent to raw material: 10:1 (mL/g). All experiments were replicated six times.

Fig. 4. Effect of ethanol concentration on the extraction yields of baicalin and baicalein from Radix Scutellariae (one extraction cycle). Extraction time: 20 min; extraction temperature: 60 ◦ C for baicalin, 30 ◦ C for baicalein; ultrasound cycle: 79%; ratio of solvent to raw material: 10:1 (mL/g); mean particle size: 0.355 mm. All experiments were replicated six times.

Fig. 3 also demonstrated that the temperature played an important role in baicalin and baicalein extraction. The extraction yields of baicalin were first enhanced with the increase in extraction temperatures, reaching a maximum yield at 60 ◦ C and having no effect on yield at higher temperatures (80 ◦ C). The extraction yields of baicalin were found to increase with increasing temperature due to the increased volatility and diffusion coefficient of baicalin in aqueous ethanol, as well as increases in solubility. Furthermore, at higher temperatures, the solvent viscosity and density decreased, thus improving the mass transfer of the solvents into the plant materials and the soluble constituents into the solvents. In addition, a lower temperature was beneficial for the extraction of baicalein. As illustrated in Fig. 3, when the extraction temperature increased from 30 to 80 ◦ C, the difference in the extraction yield of baicalein decreased slightly to 17.26%. In particular, we found that the best temperature was 30 ◦ C, the lowest temperature tested. To date, the effect of temperature on baicalein extraction under UAE has not been well studied. We assumed that some decomposition of baicalein occurred at these higher temperatures in the extraction vessel. However, the effect of extraction temperature on the extraction yield in the literature was inconsistent. Ma et al. (2008b) found that ultrasound did not result in any degradation of hesperidin in penggan (Citrus reticulata) peel at 40 ◦ C for 160 min. Similar results have also been shown by Khan et al. (2010). Ma et al. (2008a), on the other hand, found that ultrasonic treatment resulted in significant degradation of phenolics from satsuma mandarin (Citrus unshiu Marc.) peels, whereas flavanone degradation under sonication was not found. Furthermore, the variation of extraction yields of target compounds with temperature under UAE may be attributed to the combination of the thermal effect and cavitation effect. By the cavitation effect, an increase in temperature would have a negative effect because the cavitation intensity decreases with increasing temperature. Therefore, the cavitation effect would make the process less efficient, and the yield of baicalein would decrease slightly at these higher temperatures (Lou et al., 2010). From the experimental results, it can be concluded that extraction temperatures of 60 and 30 ◦ C are the most appropriate for extraction of baicalin and baicalein, respectively, from Radix Scutellariae.

were performed for 20 min at a ratio of solvent to raw material of 10:1 (mL/g), an ultrasound cycle of 79% and mean material particle size of 0.355 mm. In addition, the extraction temperatures of baicalin and baicalein were 60 and 30 ◦ C, respectively. Ethanol–water mixtures were found to be more effective for the extraction of baicalin and baicalein than absolute ethanol or water as shown in Fig. 4 (one extraction cycle). Similar observations of greater increase in extraction efficiency as a result of UAE in aqueous ethanol compared with absolute ethanol were reported previously (Boonkird et al., 2008). The increase in the extractability of baicalin and baicalein with the introduction of water to ethanol can be attributed to the increase in permeability of plant tissues in the presence of water in extracting solvents, which enables better mass transfer by diffusion. In the presence of water, the intensity of ultrasonic cavitation in the mixed solvent system of ethanol and water was further increased as the surface tension increased, and the viscosity and the vapor pressure decreased (Rostagno et al., 2003). In addition to cavitation effects, the increase in extraction yields could be explained by the sound absorption property of an ethanol–water solution. However, beyond a certain water-content threshold, the extraction yield starts to decrease. Fig. 4 shows that once the concentration of ethanol was increased from 0 to 40%, the extraction yield of baicalin decreased, indicating an optimum ethanol concentration in the range of 40%. For baicalein, the most efficient extraction condition was at 50% ethanol, a difference that may be due to the differing water concentrations. When the water concentration was above the threshold, ethanol solutions were not effective for the extraction of baicalin and baicalein due to the increased polarity of the mixture. Baicalin and baicalein also have very poor solubilities in water, thus limiting their quantities inside the extracts. Therefore, an increase in water concentrations results in opposing forces, and it is reasonable to expect that an optimum concentration of ethanol may exist. This proposal is in agreement with earlier studies (Yang et al., 2012; Yang et al., in press). As can be inferred from the results, 40% ethanol in water was found to be optimum for the extraction of baicalin from Radix Scutellariae, and 50% ethanol for the extraction of baicalein from Radix Scutellariae (Fig. 4).

3.2.2. Effect of ethanol concentration The experiments studying the effect of various concentrations of ethanol on the extraction yields of baicalin and baicalein

3.2.3. Effect of extraction time To achieve high recoveries, a primary extraction step under stirring (5 min) was performed; this step should allow for better

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Fig. 5. Effect of extraction time on the extraction yields of baicalin and baicalein from Radix Scutellariae (one extraction cycle). Extraction temperature: 60 ◦ C for baicalin, 30 ◦ C for baicalein; ultrasound cycle: 79%; ethanol concentration: 40% (v/v) for baicalin, 50% (v/v) for baicalein; mean particle size: 0.355 mm; ratio of solvent to raw material: 10:1 (mL/g). All experiments were replicated six times.

Fig. 6. Effect of the ratio of solvent to raw material on the extraction yields of baicalin and baicalein from Radix Scutellariae (one extraction cycle). Extraction time: 20 min; extraction temperature: 60 ◦ C for baicalin, 30 ◦ C for baicalein; ultrasound cycle: 79%; ethanol concentration: 40% (v/v) for baicalin, 50% (v/v) for baicalein; mean particle size: 0.355 mm. All experiments were replicated six times.

penetration of the solvent in the matrix. This step was followed by ultrasonic extraction to enhance analyte solubility in the extraction medium. To determine the suitable extraction time for baicalin and baicalein extraction with UAE, the extraction time was varied from 5 to 60 min (one extraction cycle). Fig. 5 shows the effect of extraction time on the extraction yields of baicalin and baicalein at a ratio of solvent to raw material of 10:1 (mL/g) and a mean material particle size of 0.355 mm using UAE at an ultrasound cycle of 79%. The extraction temperatures of baicalin and baicalein were 60 and 30 ◦ C, respectively, and the ethanol concentration for the extraction of baicalin and baicalein were 40 and 50%, respectively. When the time was extended, the extraction yields of baicalin and baicalein slightly increased, especially during the 10-min extraction. However, there was no further improvement after 20 min of extraction. Compared with HRE (Table 1), UAE significantly shortened the extraction time and improved the extraction yields of target compounds. It is thought that swelling and hydration could be accelerated by the cavitation effects of the ultrasonic waves, thus resulting in a probable enlargement of the pores of the cell walls. Furthermore, better diffusion through the plant cell walls, disruption and washing out of the cell contents were also thought to facilitate extraction performance. In addition, the corresponding reduction in the size of the material’s particles by ultrasound disintegration will increase the number of cells directly exposed to extraction by the solvent and ultrasonic cavitation (Vinatoru, 2001). The cavitation effects further caused tissue disruption and a good penetration of the solvent into the tissue matrix (Sun and Tomkinson, 2002). Therefore, the analytes of interest are more easily released from the plant tissues into the extraction medium, thereby boosting yields in a relevantly shorter time. It is evident from Fig. 5 that the baicalin and baicalein extraction yield increases rapidly with the extraction time for the first 10 min and slowly in the next 20 min and varies very little with further increase in the time. The rate of baicalin and baicalein extraction was high during the first 10 min due to the large difference between the initial baicalin and baicalein concentration in the extraction medium and their solubility. Another reason for the initially high rate could be that baicalin and baicalein located in the outer region of particle were more readily accessible than those in the inner part where the tissue matrix is more intact. The extraction from the outer part was attributed to an external mass transfer, which was convective in this case because fluid motion was provided as

a result of the cavitation effects of ultrasonic waves. At later times, baicalin and baicalein from the inner part of the root particles must diffuse through the pores of the root materials, resulting in much slower extraction rates. The obtained results suggest that under the tested conditions, an extraction time of 20 min is the most suitable time to extract baicalin and baicalein from Radix Scutellariae. 3.2.4. Effect of the ratio of solvent to raw material In general, a larger solvent volume can dissolve constituents more effectively, leading to an improvement of the extraction yields of target compounds. However, the effect of the ratio of solvent to raw material on extraction yield in the literature was inconsistent. Previous studies showed that the large ratio of solvent to raw material resulted in a decrease of extraction yield (Xia et al., 2011). From an economic perspective, using a large amount of solvent was not considered cost-effective due to the high operating cost of solvents and energy consumption. Fig. 6 shows the influence of the ratio of solvent to raw material on the extraction of baicalin and baicalein (one extraction cycle). The experiments were carried out in ratios of solvent to raw material ranging from 6 to 22 mL/g with a mean material particle size of 0.355 mm, an ultrasound cycle of 79%, an extraction time of 20 min, an extraction temperature of 60 ◦ C (for baicalin) or 30 ◦ C (for baicalein) and an ethanol concentration of 40% (for baicalin) or 50% (for baicalein). The results indicate that the increase in extraction yield is more pronounced when the ratio of solvent to raw material increases from 6 to 10 mL/g. This increase is flattened when the ratio of solvent to raw material is more than 12 mL/g. Therefore, considering the yields of both baicalin and baicalein, the ratio of solvent to raw material of 10 mL/g was selected. 3.2.5. Effect of ultrasound cycle Recent studies have shown that UAE enhances extraction efficiency by increasing the yield and shortening the time of extraction of target compounds from various plant tissues. From previous studies (Sun et al., 2011), the ultrasound cycle was considered to be an important factor affecting the efficiency of target compound extraction from raw materials. The results indicate that the proper use of a pulse mode of ultrasound (intermittent sonication) can replace continuous irradiation by ultrasound and thus obtain better extraction yields from raw materials and reduce overall energy consumption. However, the effect of ultrasound cycle on extraction

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Y.-C. Yang et al. / Industrial Crops and Products 45 (2013) 182–190 Table 2 Repeatability and reproducibility of the developed UAE–HPLC method. Analytes

Baicalin Baicalein

Repeatabilitya

Reproducibilityb c

Mean (mg/g)

RSD (%)

Mean (mg/g)

RSD (%)c

74.4585 20.0578

3.74 4.74

73.8972 19.5792

6.39 7.02

a Six samples of RS1 from the same source were extracted and analyzed under the optimum conditions during one day. b The extraction yields obtained from six independent extractions (RS1) carried out on five consecutive days. c RSD (%) = (SD/mean) × 100.

Fig. 7. Effect of ultrasound cycle on the extraction yields of baicalin and baicalein from Radix Scutellariae (one extraction cycle). Extraction time: 20 min; extraction temperature: 60 ◦ C for baicalin, 30 ◦ C for baicalein; ethanol concentration: 40% (v/v) for baicalin, 50% (v/v) for baicalein; mean particle size: 0.355 mm; ratio of solvent to raw material: 10:1 (mL/g). All experiments were replicated six times.

performances in the literature was inconsistent. This result was not in agreement with earlier studies (Luque-García and Luque de Castro, 2004). To achieve a relatively complete extraction of baicalin and baicalein from Radix Scutellariae by UAE, the ultrasound cycle was increased from 0 to 100% to study the effect of the ultrasound cycle using the above optimized conditions (solvent to raw material: 10 mL/g; mean material particle size: 0.355 mm; extraction time: 20 min; extraction temperature: 60 ◦ C (for baicalin) or 30 ◦ C (for baicalein); ethanol concentration: 40% (for baicalin) or 50% (for baicalein). According to the results (Fig. 7), the extraction yield was enhanced by increasing the ultrasound cycle. However, because the difference between the obtained extraction yields of 79 and 100% was not significantly different, the ultrasound cycle used to obtain a 79% yield is selected as optimal condition because it requires less electrical energy. 3.3. Repeatability and reproducibility of the combined UAE-HPLC method To evaluate the repeatability of the developed UAE and HPLC methods (UAE–HPLC), six different working solutions prepared

from the same sample (5.0 g, Radix Scutellariae, RS1) were analyzed. The relative standard deviation (RSD, %) values were calculated as a measurement of method repeatability. The extraction yields of baicalin and baicalein demonstrated good repeatability (Table 2). It could also be observed that repeatability in term of RSD were less than 4.75%, which demonstrated that the values were all within an acceptable range and that the method was accurate and precise. Furthermore, the reproducibility was evaluated by calculating the extraction yields obtained from six independent extractions carried out on five consecutive days. The extraction yields (reproducibility) of baicalin and baicalein were 73.8972 ± 4.7220 mg/g and 19.5792 ± 1.3745 mg/g, respectively (n = 30). The RSD of the two compounds were less than 7.03% (Table 2). The reproducibility of this method was satisfactory. 3.4. Comparison between HRE and UAE The extraction recoveries of baicalin and baicalein obtained by UAE were compared to those obtained by HRE, as shown in Table 3. Optimized conditions determined from the above experiments were applied to the UAE and HRE experiments. Each experiment was replicated six times for each 5 g Radix Scutellariae sample, either by UAE or HRE. The mean extraction recoveries for UAE and HRE were 52.31 and 37.22% for baicalein and 67.04 and 55.17% for baicalin (one extraction cycle), respectively. The mean extraction recoveries of baicalin and baicalein by UAE were considerably higher than those obtained by HRE. The mean extraction recoveries reach 100% after 3 extraction cycles using UAE in the present work. After three extraction cycles, the highest yield of baicalin and baicalein by UAE under optimum extraction conditions were

Table 3 Comparison of extraction recoveries and extraction conditions obtained by the UAE and HRE methods. Parameters

Herbal sample Material’s mean particle size (mm) Plant weight (g) Ultrasonic frequency (kHz) Ultrasound cycle (%) Stirring rate (rpm) Extraction time (min) Liquid/solid ratio (mL/g) Extraction temperature (◦ C): Baicalin: Baicalein: Ethanol (%)a : Baicalin: Baicalein: Extraction cycles: Mean recovery (%)b : Baicalin: Baicalein: a b

Extraction mode HRE

UAE

RS1 0.355 5 – – 200 60 14

RS1 0.355 5 40 79 200 20 10

80 ◦ C 40 ◦ C

60 ◦ C 30 ◦ C

50% 90% 1

2

3

4

40% 50% 1

2

3

4

55.17 37.22

78.57 68.94

95.92 89.15

100.11 99.98

67.04 52.31

84.57 79.83

100.08 99.99

– –

Ethanol concentration in water (v/v, %) Recovery (%) = [(found value–content/added value)] × 100

Y.-C. Yang et al. / Industrial Crops and Products 45 (2013) 182–190

110.3167 ± 4.1258 and 37.4254 ± 1.7665 mg/g, respectively. HRE could not produce the same level of recovery (100%) until after 4 extraction cycles. Thus, it can be concluded that the UAE procedure proposed here offers a more efficient target compound isolation method compared to classical processes. This conclusion is consistent with UAE having the potential to extract target compounds from raw materials in better yields than conventional techniques (Boonkird et al., 2008). At the extraction temperature of 60 ◦ C, ultrasound with 40% ethanol for 20 min gave the highest recovery of baicalin (67.04%). This result was comparable to that obtained from HRE in 50% ethanol at 80 ◦ C for 60 min. Clearly, by reducing the time required and the temperature for extraction, UAE was shown to be a promising method that offers improved extraction efficiency. In addition, using 50% ethanol as solvent (30 ◦ C) in UAE could increase the recovery of baicalein up to 52.31%, which was comparable to using HRE in 90% ethanol (40 ◦ C) but with shorter extraction times. To sum up, UAE improves the extraction efficiency of baicalin and baicalein from Radix Scutellariae. Compared with HRE, UAE is also much more economical in terms of time, solvent use, temperature requirements and energy consumption. 4. Conclusion UAE, an alternative method for the extraction of baicalin and baicalein from Radix Scutellariae, has been investigated. The obtained crude extract yields were also compared with HRE in order to select the best operation parameters. On the basis of economic considerations, the optimal procedure for extracting baicalin and baicalein from Radix Scutellariae was at an ultrasonic frequency of 40 kHz, a power of 185 W, an ultrasound cycle of 79%, a mean material particle size of 0.355 mm, a ratio of solvent to raw material of 10:1 (mL/g) and 3 cycles. However, the optimum extraction conditions varied between flavanone compounds. For a given ultrasound cycle, mean material particle size and ratio of solvent to raw material, 40% ethanol in water was found to be optimum for the extraction of baicalin from Radix Scutellariae at 60 ◦ C for 20 min and 50% ethanol for the extraction of baicalein from Radix Scutellariae at 30 ◦ C for 20 min. The highest yield of baicalin and baicalein by UAE under optimum extraction conditions was higher than the yield obtained by HRE. Additionally, UAE showed a lower extraction time and solvent consumption at lower extraction temperatures. Therefore, this study demonstrated promising results for UAE in the isolation baicalin and baicalein from Radix Scutellariae. Acknowledgements The authors would like to thank the National Science Council of the Republic of China, Taiwan for financially supporting this research. We greatly thank Kaiser Pharmaceutical Limited Company (KPC, Tainan, Taiwan) for kindly providing and authenticating the plant materials used in this research. We are indebted to Professor Ian-Lih Tsai (Kaohsiung Medical University, Taiwan) for technical assistance. We are grateful for the editorial assistance of Miss Wei-Tzu Sun, Yu-En Chen, Wen-Tzu Lu (Kaohsiung Medical University, Taiwan) and Hsiang-hung Wei (National Hsinchu University, Taiwan). Finally, the authors would like to acknowledge the anonymous editor and referees for their constructive comments. References Biesaga, M., 2011. Influence of extraction methods on stability of flavonoids. J. Chromatogr. A 1218, 2505–2512. Boonkird, S., Phisalaphong, C., Phisalaphong, M., 2008. Ultrasound-assisted extraction of capsaicinoids from Capsicum frutescens on a lab- and pilot-plant scale. Ultrason. Sonochem. 15, 1075–1079. Briars, R., Paniwnyk, L., 2013. Effect of ultrasound on the extraction of artemisinin from Artemisia annua. Ind. Crop. Prod. 42, 595–600.

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