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Chemical Engineering and Processing: Process IntensificationVolume 47, Issue 12, November 2008, Pages 22562261Extraction of isoflavonoids from Pueraria by combining ultrasound with microwave vacuum Yang Hua, Tao Wanga, MingxiaoWangb, Sufang Hana, Pingyu Wana, Maohong Fanc, a Beijing University of Chemical Technology, Beijing 100029, China b General Hospital of China National Coal Group Corp., Beijing 110013, China c School of Materials Science and Engineering, Georgia Institute of Technology, Atlanta, GA 30332-0245, USA Received 24 December 2006. Revised 30 September 2007. Accepted 12 December 2007. Available online 11 January 2008. /10.1016/j.cep.2007.12.013, How to Cite or Link Using DOI Permissions & ReprintsAbstractThis study proposes a new method to quickly extract and dry isoflavonoids from Pueraria Lobata Ohwi (Pueraria) by combining ultrasound and microwave technologies. The time required to extract isoflavonoids at comparable levels of production by ultrasound disruption was 20 times shorter than by conventional reflux extraction. Moreover, test results for drying the extracted substance from Pueraria show that the microwave-vacuum method is 10 times faster than the conventional two-step vacuum approach. Finally, different instrumental analyses of isoflavonoids obtained from Pueraria using the proposed new method show that extraction by ultrasound disruption and microwave-vacuum drying affects neither the structure nor the composition of the extracted substance.Keywords：Extraction; Isoflavonoids; Microwave drying; Ultrasound1. IntroductionUltrasound has been used to extract compounds from the various parts of plants for more than three decades 1. Due to the disruption of cell walls and enhanced mass transfer of cell contents, ultrasound is able to accelerate the extraction of organic compounds from the bodies of plants 2and3. Compared with conventional solvent extraction, the use of ultrasound makes the extraction of valuable compounds more efficient by means of shorter time frames and lower extraction temperatures. Ultrasound is currently employed to extract such pharmacologically active compounds as polysaccharides, cellulose, flavonoids, saturated hydrocarbons, fatty acid esters, and steroids from plant materials 4.Microwave drying is often used to evaporate water in wood, foodstuffs, drugs, and ores, among other commodities 5and6; it can also be used for distillation 7 and extraction 8, 9and10. Combining the application of microwaves with vacuum techniques for drying offers two major advantages, namely, rapid drying due to the ability of microwaves to heat solvents instantaneously and homogeneously, and enhanced rate and extent of mass transfer at sub-atmospheric pressure and low temperatures, which is essential for thermo-labile products. Such drying technologies are therefore important for industries such as pharmaceuticals 11.The present study of the extraction of isoflavonoids from Pueraria by ultrasound disruption, together with their drying by means of combined microwave and vacuum technologies, offers an alternative to conventional technologies for extracting and drying isoflavonoids from Pueraria.2. Experimental2.1. Plant materials and chemicalsPueraria collected in Chinas Henan province was washed, dried, and cut into 5mm segments. The standard sample of Puerarin was supplied by Chinas National Institute for the Control of Pharmaceutical and Biological Products. Analytical reagent-grade ethanol and ethyl acetate were used in all experiments.2.2. ExtractionFig. 1 shows the combined microwave-ultrasound experimental setup used for extraction. The apparatus consists mainly of an extraction component (a), a microwave drying component (b) and a solvent recycling component (c). Ultrasound extraction was carried out using a CF-1520 50kHz 1200W ultrasound disintegrator. A GALANZ WP700L17 microwave oven with a Teflon evaporating dish was used for drying.Fig. 1.Schematic drawing of the apparatus used for extraction (a: extracting part; b: microwave drying part; c: solvent recycling part; 1: bracket; 2: ultrasound transducer; 3: beaker; 4: sealing gasket; 5: teflon evaporating dish; 6: turning plate; 7: microwave oven; 8: teflon tube; 9: latex tube; 10: inlet; 11: outlet; 12: vacuum sucker).View thumbnail imagesTwenty grams of Pueraria were put into a 1000ml beaker for ultrasound extraction. After 400ml of 70% ethanol added, the beaker was put on the ultrasound disintegrator, followed by extraction under the ultrasound horn for 30min at room temperature. Samples taken periodically were analyzed using an UV-2102PC UltravioletVisible (UVVIS) spectrophotometer.A reflux extraction experiment was also performed for purposes of comparison. After 20g of Pueraria were transferred into a 1000ml three-neck flask, 400ml of 70% ethanol was added. The mixture was stirred at 600rpm and refluxed for 120min, during which samples were taken periodically for analysis by UV spectrophotometer.2.3. Drying of extracted productA GALANZ WP700L17 microwave oven with a Teflon evaporating dish was used for drying the extracted product. For recycling the solvent and ensuring safety, the microwave oven was made airproof by drilling a hole through its roof and passing a Teflon tube through the hole. The tube was connected to a vacuum system to withdraw steam and volatile chemicals, allowing water and organic solvents to be evaporated by the microwave-vacuum apparatus.To initiate drying, 200ml of ultrasound-extracted resultant was put in an evaporating dish and placed in the microwave oven. The extracted resultant became solid in about 10min. Another 200ml of extracted resultant was dried using a rotating evaporator until no distillate came out, followed by drying it into powders for about 80min in an oven.2.4. Analytical methodsThe extracted Pueraria isoflavonoids were analyzed by UV at 250nm wavelength 12. The standard curve of absorbance versus Puerarin concentration was obtained in the following manner: ten milligrams standard sample of Puerarin was first put into a 50ml volumetric flask; then 95% ethanol was added to the scale, followed by shaking the mixture until it was homogeneous. Aliquots of solutions (0.2, 0.4, 0.6, 0.8, and 1.0ml) were taken from the flask and placed into 10ml volumetric flasks. 1.0ml of ethanol was then added to each flask and deionized water was added to the flask scales to complete the preparation of standard solutions. Blank samples were prepared using the same procedures previously mentioned but without the addition of extracted isoflavonoids. The UV absorbance of blank and each standard sample was measured at a 250nm wavelength.The extract resultant was analyzed as follows; one milliliter of extract was transferred into a 50ml volumetric flask followed by the addition of 95% ethanol to the scale and mixing. The solution was left overnight. One milliliter of supernatant was then transferred into a 25ml volumetric flask; deionized water was added to the scale, followed by measuring the absorbance at 250nm. The concentration of isoflavonoids in the solution was obtained from the standard curve. The level of isoflavonoids in the sample is calculated as (1)where c1 is the concentration of isoflavonoids in the extract resultant, V1 is the volume of extract resultant, and mpueraria is the weight of the Pueraria. Twenty milligrams of extracted product (powder) and 30ml of 95% ethanol were mixed in a 50ml volumetric flask to initiate analysis of the extract. The mixture was then heated in a hot water bath to dissolve the product. After cooling, the flask was filled with 95% ethanol to the scale. After about 12h, 1.0ml supernatant was placed in a 25ml volumetric flask and diluted with deionized water to the scale. The absorbance of the solution was then measured at 250nm. The yield of powder, content of total isoflavonoids in powder, and total yield of isoflavonoids are defined as follows: (2)(3)(4)where mpowder and mpueraria are the weights of the powder and Pueraria, respectively, c2 is concentration of the sample solution, and V2 is the dilution volume. An S-250 scanning electron microscope was used to prove the cavitations and hitting effect of ultrasound disruption on the cells of Pueraria. To examine the effect of ultrasound disruption and microwave drying on the extracted isoflavonoids from Pueraria, the extracted resultant was analyzed with a VECIDR22 Infrared (IR) Spectrometer and by LC5500 High Performance Liquid Chromatography (HPLC). The HPLC was operated with a UV detector (wavelength set at 250nm) and a C18 chromatography column. Methanolwater (3:7, v/v) was used as the mobile phase at the flow rate of 1mlmin1 and a 0.02ml sample was injected.3. Results and discussion3.1. Choice of extracting conditions of ultrasoundDifferent solvents, including water, methanol, ethanol, and ethyl acetate, were compared for their extraction abilities. The same solvent volumes and extraction times were used in the experiments for comparative extraction tests. The test results are shown in Fig. 2. As shown, the extraction efficiency for isoflavonoids from Pueraria was significantly affected by the type of solvent used. Although water can be used to achieve the highest extraction efficiency, too much starch was dissolved in water, which made the extracted resultant turbid, ropy, and difficult in filtration. Since methanol is toxic and ethyl acetate is costly, a mixture of ethanol and water was chosen as an extraction solvent. Among the solvents tested, an ethanolwater mixture at a 7:3 volume ratio proved to have the highest extraction efficiency, as shown in Fig. 2.Fig. 2.The effect of different solvents on ultrasound based extracting efficiency of isoflavonoids (solvent volume: 400ml; liquid/solid ratio: 20ml/1g; extracting time: 20min).View thumbnail imagesThe effect of the ratio the volume of solvent (i.e. a mixture of ethanol and water at a 7:3 volume ratio) to the weight of Pueraria (liquid/solid ratio) on the extraction efficiency of isoflavonoids using ultrasound was studied, with the results listed in Table 1. As shown, the increasing liquid/solid ratio led to a considerable increase in extraction efficiency until the ratio reached 20:1, which is considered optimal for the most efficient use of solvent and energy.Table 1. Effect of the ratio of solvent volume to weight of Pueraria (liquid/solid ratio) on the ultrasound based extracting efficiency for isoflavonoidsLiquid/solid ratio101520253040Content of total isoflavonoids (%)11.715.517.217.517.918.1Full-size table3.2. Comparison between ultrasound-disrupting extraction and refluxing extractionThe effect of time on the efficiency of isoflavonoid extraction from Pueraria using both ultrasound disruption and reflux extraction was studied. The relationship between the levels of total isoflavonoids and extraction time is displayed in Fig. 3. As shown, the levels of isoflavonoids extracted from Pueraria by ultrasound disruption reached about 19% over 20min, whereas the levels of isoflavonoids extracted through reflux were only 14.3% and 16.5%, after 1 and 2h, respectively. By contrast, ultrasound disruption took only 5min to reach a comparable 16.0% level of extraction efficiency (Fig. 3), about one-twentieth the time required by reflux extraction, clearly demonstrating that ultrasound disruption is superior to reflux extraction.Fig. 3.Comparison between ultrasound-disrupting and refluxing extraction for isoflavonoids (weight of Pueraria: 20g; extraction solvent: 400ml of 70% ethanol; liquid/solid ratio: 20ml/1g).View thumbnail imagesIt is well known that most biologically active compounds of plants exist in their cell walls. To extract these compounds, the cell walls can be disrupted by ultrasound extraction. Ultrasound causes intense shaking, high acceleration, intense cavitations, and stirring, all of which can accelerate the dissolution of pharmacological agents. Furthermore, enhancement of the extraction rate shortens extraction time, thereby conserving solvent and mitigating the effects of high temperatures on the effectiveness of pharmacological agents. In order to confirm the cavitations and hitting effect of ultrasound disruption on cells of Pueraria, scanning electron microscopy (SEM) was used to characterize Pueraria cells before and after both ultrasound disruption and reflux extraction. Fig. 4 shows the profiles of cells, arranged in order, with solid substances apparent in dry Pueraria cells (Fig. 4a). Some small crannies were observed in a few cells and no solid substances were shown in Pueraria cells after 2h of reflux extraction (Fig. 4b). This can be explained by the fact that the solvent used during extraction entered the cells through their gaps and crannies and contacted the solid substances, consequently moving their pharmacological compounds into the solvent. By contrast, after 20min of ultrasound, the cells could not be distinguished and their walls were almost cracked and disrupted, as seen in Fig. 4c, which resulted from the hitting and cavitating of ultrasounds intense shaking. The huge instantaneous energy generated by the ultrasound system can lead to the disruption of Pueraria cells and the quick dissolution of the isoflavonoids in Pueraria cells in solvent without a permeation process.Fig. 4.Scanning electron microscopy (SEM) photos of Pueraria cells (a: dry Pueraria cells; b: Pueraria cells obtained through refluxing for 2h; c: Pueraria cells obtained through the action of ultrasound for 20min; amplification magnitude: 200,000; electron accelerating voltage: 19kV).View thumbnail imagesIsoflavonoids in Pueraria are composed primarily of puerarin, daidzin, and daidzein 13. The structures of the three components are shown in Fig. 5. To examine the effects of ultrasound disruption on the extracted isoflavonoids from Pueraria, the extracted resultant was analyzed by HPLC, UV Spec, and IR Spec, with the results presented in Fig. 6. Fig. 6a and b are, respectively, the IR spectra of the powder extracted by ultrasound disruption for 20min and by refluxing for 2h with 70% ethanol as solvent. Fig. 6 shows that the locations of characteristic functional groups of the two samples are identical in their IR spectra, which demonstrates that extraction by ultrasound disruption generates no negative effects on the structure and components of extracted resultants compared to the conventional reflux extraction method. The UV spectra for the standard Puerarin solutions and extracted resultants from ultrasound disruption and refluxing are shown in Fig. 7. No significant differences were shown among the three curves, except for the heights of absorption peaks at 250nm. The retention times in HPLC chromatograms for the resultants from standard Puerarin samples extracted by ultrasound disruption and refluxing were 11.38, 11.29, and 11.30min, respectively. The contents of extracted resultants from ultrasound disruption and refluxing under the given conditions were, correspondingly, 3.6% and 3.7%, which indicates that Puerarin was not destroyed by ultrasound.Fig. 5.The molecular structures of isoflavonoids in Pueraria.View thumbnail imagesFig. 6.IR spectra of resultant extracted by ultrasound (a) and by refluxing (b) (refluxing extraction time: 2h; ultrasound disruption time: 20min; wavenumber scan range: 4000500cm1; weight of Pueraria: 20g; extraction solvent: 400ml of 70% ethanol; liquid/solid ratio: 20ml/1g; drying method: vacuum).View thumbnail imagesFig. 7.UV spectra of standard sample (a), resultant extracted by ultrasound (b) and resultant extracted by refluxing (c) (wavelength scan range: 200600nm; ultrasound disruption time: 20min; refluxing extraction time: 2h; weight of Pueraria: 20g; extraction solvent: 400ml of 70% ethanol; liquid/solid ratio: 20ml/1g).View thumbnail images3.3. Comparison between microwave and conventional drying methodsThe results of microwave and conventional drying of 200ml of ultrasound-extracted resultants are listed in Table 2. It can be seen that the rate of microwave-vacuum drying is 11 times faster than that of conventional vacuum drying, even though the yields of total isoflavonoids were similar.Table 2. Comparison between microwave and conventional drying methodsDrying methodTime of drying (min)Yield of the powder (%)Content of total isoflavonoids in the powder (%)Yield of total isoflavonoids (%)Conventional vacuum drying14033.348.216.0Microwave vacuum drying1330.850.115.4Extraction method: ultrasound extraction; extracting temperature: room temperature; extraction solvent: 400ml of 70% ethanol; extracting time: 20min.Full-size tableThe products obtained from 5.1% Puerarin with ultrasound-disrupting extraction and microwave-vacuum drying, and those from 4.8% Puerarin obtained with ultrasound-disrupting extraction and conventional vacuum drying were characterized by HPLC. The similarity of retention times (11.33 and 11.20min, respectively) demonstrates that Puerarin isoflavonoids were not destroyed by microwave-vacuum drying, which can be further proven by the similarity of their IR spectra as shown in Fig. 8.Fig. 8.IR spectra of products obtained by ultrasound-disrupting extraction and microwave vacuum (a) and ultrasound-disrupting extraction as well as conventional vacuum drying (b) (drying time of microwave vacuum: 10min; drying time of conventional vacuum: 80min; wavenumber scan range: 4000500cm1).View thumbnail images4. ConclusionsThe cell walls of Pueraria can be disrupted by ultrasound. The extraction time of isoflavonoids from Puera