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Ind. Eng. Chem. Res. 2007, 46, 6584-6589
Application of Mechanochemical Pretreatment to Aqueous Extraction of Isofraxidin from Eleutherococcus Senticosus Ying Liu,† Li-Ji Jin,†,‡ Xiao-Yu Li,† and Yong-Ping Xu*,†,‡ Department of Bioscience and Biotechnology, Dalian UniVersity of Technology, Dalian 116024, China, and State Key Laboratory of Fine Chemicals, Dalian UniVersity of Technology, Dalian 116012, China
A mechanochemical pretreatment (MCPT) was applied to aqueous extraction of isofraxidin from the Asian herb Eleutherococcus senticosus. Six extraction variables (Na2CO3 content, particle size, solvent, extraction time, liquid/solid ratio, and extraction temperature) were varied in order to optimize extraction efficiency. Optimal extraction was obtained using superfine powdered plant material (D95 e 44 µm) with Na2CO3 content of 2.0% (w/w) and extraction with water (liquid/solid ratio of 20:1, mL/g) for 5 min at 25 °C. The efficiency of mechanochemical-assisted extraction (MCAE) of isofraxidin was compared to that of heat-reflux and superfine grinding extraction. With the optimized parameter settings, MCAE with water as solvent gave the highest isofraxidin yield while reducing both extraction time and energy costs compared to the conventional methods. 1. Introduction Plant secondary metabolites constitute a biologically and chemically diverse group of substances with a broad range of applications, including fragrances, spices, and medicines. Past methods for extracting these bioactive compounds from complex plant matrixes have varied from traditional solvent extractions to more modern techniques such as supercritical fluid extractions,1,2 pressurized fluids extraction,3 microwave-assisted extraction,4,5 and ultrasound-assisted extraction.6,7 In developing these methods, researchers have sought to maintain or improve yield while addressing limitations such as degradation of labile or volatile compounds during lengthy extraction periods at high temperatures, requirements for hazardous organic solvents, expensive equipment, strong analyte-matrix interactions, and high energy demands.8-10 In the past decade, advances in mechanical devices have enabled development of mechanochemistry as an alternative extraction procedure. This branch of chemistry deals with the chemical and physicochemical changes of substances in all states of aggregation, e.g., such as occur with the combined action of pressure and shear in energy-intensive grinding mills.11 Application of mechanochemistry as a preliminary treatment in extracting compounds from plants may represent a novel tool for production of plant products. The procedure of grinding plant raw material in the presence of a solid reagent combines increased total contact surface area with destruction of the cell wall. Mechanochemical pretreatment (MCPT) also allows chemical transformation of target substance(s) to improve the water solubility.12 The technique has improved aqueous extraction yield of triterpene acids from fir needles by 35.9%12 and extraction of phytoecdysteroids from Serratula coronata L. by 103.7%.13 A preliminary investigation in our laboratory determined that MCPT significantly increased the yield of bioactive compounds from two herbs, Eleutherococcus senticosus and Crataegus * Corresponding author. Tel.: +86-411-8471-9811. Fax: +86-4118470-6359. E-mail:
[email protected]. † Department of Bioscience and Biotechnology, Dalian University of Technology. ‡ State Key Laboratory of Fine Chemicals, Dalian University of Technology.
Figure 1. Molecular structure of isofraxidin (7-hydroxy-6,8-dimethoxycoumarin).
pinnatifida, as compared with heat-reflux extraction. This novel application of mechanochemical-assisted extraction (MCAE) proved to be an efficient, rapid extraction technique, requiring only the simple process of stirring with water as the only solvent. With an extraction period of 0.05) beyond D95 e 44 µm. Thus, considering the longer treatment period and higher energy consumption required for continued processing, particle size of D95 e 44 µm was selected as the standard condition. 3.4. Effect of Extraction Solvent. Solvent choice is dictated by the solubility of interest analytes and the interaction between solvent and matrix.17 Simple coumarins like isofraxidin are fully
soluble in methanol, ethanol, and aqueous-alkaline solvent, are partially soluble in hot water, and are insoluble in cold water (at room temperature).18 Four solvents, water, 40%, 60%, and 100% (v/v) aqueous ethanol, were selected for study on the basis of current practice. Aqueous ethanol is a relatively good solvent for many natural compounds in standard extraction techniques.19-21 Water, a polar solvent, has not typically been sufficient to give good yield under conventional conditions, but it was included as an option for its ready availability, low cost, and ease of waste management. To compare extraction solvents, plant materials from treatments M and MC were extracted using Procedure 1 (vibrational mill WZJ(BFM)-6J), and the control material (CON) was extracted by heat-reflux. All four solvents were studied under the same extraction conditions: the defined standard conditions for M and MC and a 2 h period heat-reflux for CON. Isofraxidin yield was influenced both by sample pretreatment and by solvent properties (Figure 5). Ethanol at 40 and 60% (v/v) resulted in the greatest yields from CON and M (averaging 0.300 and 0.387 mg/g of dry material, respectively), and water resulted in the lowest yields (0.115 and 0.174 mg/g of dry material, respectively), which is consistent with conventional practice. With treatment MC, however, the yield was highest (p < 0.05) with water, compared to the ethanol solvents, and at 0.481 mg/g, it was also higher (p < 0.05) than that from other treatment × solvent combinations. Without MCPT, water was an inadequate solvent for extracting isofraxidin. Novel mechanochemical activation with Na2CO3, however, increased the water solubility of the compound, enabling water to be used as an effective solvent for extraction. On the basis of these observations, water was selected as the standard extraction solvent for MCAE, and 60% (v/v) aqueous ethanol was selected as the standard extraction solvent for CON and M (no chemicals) treatments. 3.5. Effect of Extraction Time. Isofraxidin yield using Procedures 1 (for M and MC) and 2 (for CON) was assessed after 2 min, 3 min, 5 min, 10 min, 20 min, 1 h, 2 h, 3 h, and 6 h. Optimal extraction times were greatly influenced by MCPT (Figure 6). With treatments M and MC, maximum yield was attained within 5 min, and it did not increase appreciably (p > 0.05) over the next 6 h. In contrast, CON samples (no vibrational grinding) required at least 3-6 h to approach good isofraxidin yield comparable to M. These results likely reflect the impact of exposure of internal cell contents to the extraction environment once grinding has broken the cell walls and eliminated their physical protection of intracellular components. Ensuring optimal extraction parameters (chemical, particle size, and solvent) will allow maximum utilization of the advantage afforded by superfine plant powder in exposing cell content. The yield from CON was comparable to that from M at 6 h, but both were lower (p < 0.05) than that from treatment MC. For these observations, 5 min of extraction time was selected as the standard condition for MCAE of isofraxidin from E. senticosus.
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Ind. Eng. Chem. Res., Vol. 46, No. 20, 2007
Table 2. Comparing MCAE with Heat-Reflux and Superfine Grinding Extractionsa MCAE
superfine grinding extraction
pretreatment extraction procedure extraction time total handing time
MC (MCAE) stirring 5 min 1h
M + Na2CO3 stirring 5 min 1h
solvent
water
water
liquid/solide ratio isofraxidin yield (mg/g of dry material)
20:1 mL/g 0.482 ( 0.018 b
20:1 mL/g 0.416 ( 0.012 a
b
Mc stirring 5 min 1h 60% (v/v) aqueous ethanol 20:1 mL/g 0.403 ( 0.013 a
heat-reflux extraction CONd heat-reflux 6h 7h 60% (v/v) aqueous ethanol 50:1 mL/g 0.424 ( 0.019 a
a Data are presented as mean ( SD (n ) 3). Means of isofraxidin yield without the same letters are significantly different (p < 0.05). b Superfine grounded material with a D95 e 44 µm, Na2CO3 content (w/w) of 2.0%, D95 e 44 µm. c Superfine grounded material with a D95 e 44 µm. d Plant material grounded by laboratory pulverizer and passed through a 0.25 mm aperture sieve. e On the basis of dry weight of E. senticosus raw material.
3.6. Effect of Liquid/Solid Ratio (mL/g) and Extraction Temperature. The effects of liquid/solid ratio and extraction temperature in the MCAE process were investigated in a similar manner to those described above for other extraction variables. The effects of varying liquid/solid ratio (mL/g) from 10:1 to 100:1 and temperature from 15 to 60 °C on extraction efficiency with the other variables standardized were less pronounced (data not shown). These results indicate that solubility is not a limiting factor under the conditions investigated. Considering the difficulty of evaporating solvent with higher liquid/solid ratio and no need to alter temperature to improve yield, 20:1 mL/g and 25 °C were selected as the standard conditions for this MCAE. 3.7. Analysis of Target Compound in MC Extract by Mass Spectrometry. Positive ion MS analysis of the target compound isolated from MC extract by semipreparative HPLC is shown in Figure 7. The major ion m/z 223 corresponding to the pseudomolecular ion of [M + H]+ and the peaks at m/z 245.1, 467.0 corresponding to [M + Na]+ and [2M + Na]+, respectively, were observed in the product ion mass spectrum of protonated compound. The MS analytical results are consistent with the standard isofraxidin with M ) 222, indicating that isofraxidin exhibits good structural stability during the MCAE procedure and keeps its therapeutic power after the extraction. 3.8. Recovery of Added Isofraxidin. The efficacy of the MCAE method was determined by spiking subsamples of previously analyzed MC samples with known amounts of pure isofraxidin (0.02, 0.04, and 0.08 mg/g) before extraction using the standard optimal conditions as selected above. The recovery of isofraxidin ranged from 81.0% to 85.5% (Table 1). Since quantitative recoveries are observed, the matrix effect does not appear to be an issue at the MCAE optimal conditions. 3.9. Comparing MCAE with Heat-Reflux and Superfine Grinding Extraction. Conventional extraction of acidic compound with aqueous-alkaline solvent has been well-studied.22,23 To investigate the unique mechanical and chemical advantages of MCAE over conventional extraction, a final investigation, besides the heat-reflux and superfine grinding extraction, was conducted in which Na2CO3 was included, but not in conjunction with the mechanical grinding processing of the E. senticosus material. Thus, four processes were compared: (i) MCAE with the optimized parameter settings determined herein (i.e., treatment MC); (ii) superfine E. senticosus particles (D95 e 44 mm, as above), but with physical mixing of the powder with Na2CO3 (D95 e 44 mm) to 2.0% (w/w) after grinding and extraction with water for 5 min; (iii) superfine grinding extraction: using vibrational mill WZJ(BFM)-6J to produce superfine E. senticosus powder with D95 e 44 mm particle size (i.e., treatment M), which is then extracted with 60% (v/v) aqueous ethanol for 5 min; and (iv) conventional heat-reflux extraction: 6 h, 60% (v/v) ethanol as solvent (i.e., treatment CON). Superfine grinding with no Na2CO3 produced comparable (p > 0.05) yield of isofraxidin (0.403 vs 0.424 mg/g) to traditional
heat-reflux extraction and enabled reduced extraction time and solvent volume (Table 2). The MCAE process defined in this study increased (p 0.05) yield, giving a yield of 13.7% lower (p
