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Microwave Multi-Stage Countercurrent Extraction of Dihydromyricetin from Ampelopsis Grossedentata

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Microwave Multi-Stage Countercurrent Extraction of Dihydromyricetin from Ampelopsis Grossedentata 374 W. LI et al.: Microwave-Assisted Extraction of Dihydromyricetin, Food Technol. Biotechnol. 45 (4) 374–380 (2007) ISSN 1330-9862 original scientific paper (FTB-1588) Microwave Multi-Stage Countercurrent Extraction of Dihydromyricetin from Ampelopsis grossedentata Wei Li1,2, Cheng Zheng3*, Jinshui Wang4, Youyuan Shao1, Yanxiang Gao2, Zhengxiang Ning4 and Yueming Jiang5 1College of Biological Engineering, Hubei University of Technology, Wuhan 430068, PR China 2College of Food Science and Nutritional Engineering, China Agriculture University, Beijing 100083, PR China 3College of Chemistry and Chemical Engineering, Guangzhou University, Guangzhou 510091, PR China 4College of Light Industry and Food Science, South China University of Technology, Guangzhou 510640, PR China 5South China Botanical Garden, Chinese Academy of Sciences, Guangzhou 510650, PR China Received: November 30, 2005 Revised version: December 4, 2006 Accepted: January 31, 2007 Summary Microwave-assisted extraction (MAE) technique in combination with multi-stage countercurrent extraction (MCE), namely microwave multi-stage countercurrent extraction (MMCE), was evaluated for the extraction of dihydromyricetin (DMY) from Ampelopsis grossedentata. Ethanol, methanol and water were used as extract solvents in the MMCE method. Of the three solvents used, water was found to be the best in extracting DMY from Ampelopsis grossedentata because it had a good extraction yield and is inexpensive, non-toxic and environmentally friendly. The optimal conditions of MMCE for the extrac- tion of DMY can be determined to be the ratio of the extraction solvent to plant material of 30:1, the extraction time of 5 min, the extraction temperature of 110 °C and the micro- wave power of 600 W. In addition, the extraction efficiency of the MMCE method was compared with that of the microwave static batch extraction (MSBE) under the optimum extraction conditions. It was found that the MMCE method offered higher extraction effi- ciency than the MSBE method. Thus, the study suggests that the MMCE method provides an alternative technique in terms of both cost and efficiency. Key words: microwave-assisted extraction, microwave multi-stage countercurrent extraction, dihydromyricetin, Ampelopsis grossedentata, extraction efficiency Introduction ducts to consumers worldwide. Microwave-assisted ex- The development of techniques for the extraction of traction (MAE) has received an increasing attention as components from medicinal plants plays an important an alternative to solid-liquid extraction for the extraction role in ensuring and providing high-quality natural pro- of secondary metabolites from plants. In contrast with *Corresponding author; Phone/Fax: ++86 20 83 500 597; E-mail: [email protected], [email protected] W. LI et al.: Microwave-Assisted Extraction of Dihydromyricetin, Food Technol. Biotechnol. 45 (4) 374–380 (2007) 375 the conventional liquid-solid extraction, the use of MAE Materials and Methods method results in significant reduction of the extraction time because the microwave heats the solvent rapidly. Ampelopsis grossedentata Furthermore, microwave can keep the temperature gra- Dried Ampelopsis grossedentata leaves produced in the dient to a minimum and accelerate the speed of heat Wuyi mountain region of China were purchased from transfer. Additionally, the MAE method allows for a sig- the Chinese Traditional Medicine Market in Guangzhou. nificant reduction in consumptions of solvents and en- The content of DMY in the dried Ampelopsis grossedentata ergy and increases extraction efficiency (1,2). Regarding the extraction, the advantage of microwave heating is was about 40 % based on the analysis by a TSP-2000A the disruption of weak hydrogen bonds promoted by high performance liquid chromatograph (HPLC, Thermo- the dipole rotation of the molecules. The migration of electric Company, USA). dissolved ions increases penetration of the solvent into the matrix and, thus, facilitates the salvation of the tar- Reagents get compounds. Microwave acts directly on water mole- The standard DMY was provided by the Laboratory cule within the cells in situ, resulting in a rapid increase of Food Chemicals at South China University of Tech- in the cell temperature. The pressure resulting from the nology. Methanol and double distilled water were of vapor of the cell leads to rupture of the cell membranes HPLC grade, while other reagents were of analytical and cell walls (3,4) and generates cavities during MAE grade. operation. This makes the internal material flow out and the solvent penetrates into the cell easily, thereby increas- DMY determination ing the extraction efficiency (5,6). DMY content was determined according to the me- Multi-stage countercurrent extraction (MCE) as a no- thod of He et al.(24), with a slight modification. Metha- vel extraction technique combines circulatory dynamic extraction and continuous countercurrent extraction (7). nol (20 mL) was added to 1.0 g of the dried Ampelopsis The MCE method offers a high extraction yield and is grossedentata. Microwave extraction was carried out using considered to save time and reduce consumption of the Microwave Accelerated Reaction System (MARS, both energy and solvent. Furthermore, the concentration CEM Corporation, Mattews, NC, USA) (300 W) with difference between the solid phase and the liquid phase closed extraction vessels. The mixture was extracted to in different extraction vessels can be maintained as con- stand for 5 min at 60 °C using a magnetic stirrer. The stant as possible during the MCE. Consequently, the use obtained mixture was filtered through filter cloth and of MCE method can increase extraction efficiency and the filtrate was then collected. The residue was extracted obtain a rapid and complete extraction of bioactive com- 6 times. All the filtrates were combined. DMY content pounds from herbs (8). was determined by HPLC on a Kromasil 5-mmC18 col- Ampelopsis grossedentata, which grows wild south of umn (250´4.60 mm). The column was eluted by a mo- the Yangtze River region in China, has been used as a bile phase consisting of a binary mixture of methanol Chinese medicinal herb for over 2000 years. Dihydromy- and water (methanol/water volume ratio=32:68), spiked ricetin (DMY), one of the flavonols (shown in Fig. 1), is with 0.1 % acetic acid as a solvent modifier. The flow a major bioactive component in Ampelopsis grossedentata rate was maintained at 1.5 mL/min. The eluent was mo- (9). DMY has a wide range of therapeutic effects and phar- nitored using a UV detection at 291 nm and the DMY macological activity, such as antioxidant ability (10–13), content was calculated according to the following for- antiviral and antibacterial properties (14–16), high UV- mula: DMY/%=[(A+11755)/38121´m]´N´V´10–3´100; -absorbing ability (17–19), cell proliferation (20) and he- where A, N and V were the sample peak area, the sam- patoprotective properties (21–23). ple dilution times, and the total volume (mL) of the ex- tract solution, respectively, while m was the mass (in mg) of Ampelopsis grossedentata. OH 3' Five-stage MMCE 4' OH 2' A five-stage MMCE was carried out according to the 1 B 8 O HO 7 method of Wang et al.(7), with some modifications. Five 1' 2 5' OH vessels were used simultaneously in the MARS. This A C 6' 3 MARS can provide 14 simultaneous extractions in Tef- 6 4 5 OH lon-lined closed vessels which allow the extraction sol- vent to be heated above the boiling point of the solvent. OH O The cover for the extraction vessel used as control had a Fig. 1. The chemical structure of DMY small hole through which a fiber-optic temperature probe was inserted to monitor the internal temperature. The MMCE was divided into the conditioning stage (the first In this study, the MAE technique in combination with phase) and the extraction stage (the second phase). In the MCE method was used to extract DMY from Ampe- the conditioning stage, the Ampelopsis grossedentata sam- lopsis grossedentata. The extraction conditions of the MMCE ple was mixed with water in each vessel and was con- technique were evaluated. In addition, the MMCE tech- tinuously extracted for a predetermined period of time. nique in terms of extraction yield and efficiency was ana- The different extraction time used for various vessels lysed in comparison with the microwave static batch ex- was predesigned to produce a systematic extract concen- traction (MSBE) method. tration along the vessel sequence. The concentration gra- 376 W. LI et al.: Microwave-Assisted Extraction of Dihydromyricetin, Food Technol. Biotechnol. 45 (4) 374–380 (2007) dients for the five-stage MMCE were obtained by ex- fective method for investigating the effects of the factor tracting the sample in vessels 1-4 for different intervals on the extraction yield by reducing the experimental (T/5, 2T/5, 3T/5 and 4T/5 respectively, where T is the numbers. Furthermore, the optimal extraction conditions total running time of the MMCE). The extraction in ves- and the significance of every factor’s effect on the yield sel 4 was transferred to vessel 5, while the extraction in can be obtained. The factor levels of the orthogonal vessel 1 was exchanged with vessel 3. Meanwhile, fresh experiments are shown in Table 1. Four factors (time, solvent and fresh sample were added to vessels 4 and 5, temperature, solvent to plant material ratio (M/S) and respectively. The MMCE was performed according to a power) of the five-stage MMCE were considered to eva- series of five steps. The duration of every step was T/5. luate the variables in the design. The experiments were Each step included four basic operations. For example, conducted three times and the data were analyzed by the first step consisted of the following operations: (i) analysis of variance (ANOVA). Water was used as sol- sample extraction for T/5; (ii) discharge of the residue vent in all orthogonal experiments. from vessel 4 and collection of the extract from vessel 5; (iii) transfer of solvent from 1®4, 2®5, 3®1 and 4®2; 4 and (iv) addition of fresh sample and fresh water to ves- Table 1.
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