Journal of Chemical Technology and Biotechnology J Chem Technol Biotechnol 80:985–991 (2005) DOI: 10.1002/jctb.1273

Glucoraphanin extraction from Cardaria draba: Part 1. Optimization of batch extraction Erin E Powell,1 Gordon A Hill,1∗ Bernhard HJ Juurlink2 and D Julie Carrier3 1Department of Chemical Engineering, University of Saskatchewan, 110 Maintenance Road, Saskatoon, SK, S7N 5C5, Canada 2Department of Anatomy and Cell Biology, University of Saskatchewan, 107 Wiggins Road, Saskatoon, SK, S7N 5E5, Canada 3Department of Biological and Agricultural Engineering, University of Arkansas, Fayetteville, AR 72701, USA

Abstract: have historically been considered an anti-nutritional component of food and feed cereal crops. Large-scale protocols have been aimed at complete elimination from plants, rather than maximizing the recovery of any particular glucosinolate compound. Recently, glucoraphanin, an alkenyl glucosinolate, has been found to have nutritional value in terms of anti-carcinogenic behavior and hypertension relief. In this work, we report on the efficient capture of glucoraphanin from the noxious weed Cardaria draba. The effect of temperature, ethanol content in the aqueous solvent, initial solvent pH, solids loading, and contact time on both glucoraphanin and glucosinalbin recovery were examined. The optimal extraction conditions, evaluated using 0.11 dm3 stirred baffled vessels, were found to be 20% aqueous ethanol solvent at 70 ◦C and an initial pH value of 3, extracted at a solid to liquid ratio of 50 g dm−3 over 20 mins. The recovery achieved with the baffled vessels was up to three times greater than the glucoraphanin yield obtained using standard analytical procedures that involved the use of 8.0 × 10−3 dm3 of hot, 80% ethanol solutions in test tubes at the same solvent loading. This corresponds to 30 mg g−1 of glucoraphanin recovered from the dried Cdrabaleaves, versus only 10 mg g−1 using the analytical method.  2005 Society of Chemical Industry

Keywords: glucoraphanin; ; extraction; optimization; Cardaria draba

NOTATION families, almost exclusively in species of the order a Radius of spherical particle (m) Capparales.1 More than 100 glucosinolates with C Concentration of solute in the solid (g m−3) different side chain structures have been described.2 CL Concentration of solute in the bulk solvent The biochemical pathway of glucosinolate synthesis (g m−3) is still partly speculative but can be considered to CO Initial concentration of solute in solid spherical be made up of three stages: amino acid side chain particle (g m−3) elongation, glucone biosynthesis from the amino acid, D Diffusion coefficient (m2 s−1) and chain modifications.2 Gls Glucosinalbin Glucoraphanin hydrolyzes to form the phase 2 Grp Glucoraphanin enzyme inducer sulforaphane that imparts numer- K Partition coefficient between solute concentra- ous health benefits. Early studies have shown tions in equilibrium in the solution and the that sulforaphane eliminates carcinogens in living particle (dimensionless) tissue.1,3 Both natural and synthetic sulforaphane r Radial location from centre in a spherical have proven to be inducers of phase 2 enzymes such particle (m) as glutathione S-transferase and quinone reductase.4 t Time (s) Juurlink5 observed that the induction of phase 2 enzymes is linked with anti-oxidant defense mecha- nisms. Experiments with spontaneously hypertensive INTRODUCTION stroke-prone rats showed that consumption of broc- Glucoraphanin is one of a group of chemically-related coli sprouts (containing 2.4 µmol glucoraphanin g−1) compounds known as glucosinolates. Glucosinolates resulted in decreased blood pressure and increased are sulfur- and nitrogen-containing compounds found endothelial cell function.6 Glucoraphanin is a sta- in vegetative and reproductive tissues of Cruciferae ble compound, whereas sulforaphane is volatile. It (syn ) and 15 other dicotyledonous plant is practical therefore to create a glucoraphanin-based

∗ Correspondence to: Gordon A Hill, Department of Chemical Engineering, University of Saskatchewan, 110 Maintenance Road, Saskatoon, SK, S7N 5C5, Canada E-mail: [email protected] Contract/grant sponsor: Natural Sciences and Engineering Research Council of Canada Contract/grant sponsor: Saskatchewan Agriculture Development Fund (Received 8 August 2004; revised version received 2 January 2005; accepted 17 January 2005) Published online 23 March 2005  2005 Society of Chemical Industry. J Chem Technol Biotechnol 0268–2575/2005/$30.00 985 EE Powell et al nutraceutical while retaining the health advantages of temperature, solvent composition (limited to ethanol sulforaphane. and water), initial solvent pH and solids loading. The physical and chemical properties of gluco- are important to its extraction from plant 7 material. Dunford and Temelli found that gluco- MATERIALS AND METHODS ◦ raphanin is thermally stable at least to 100 C. Glu- Plant material coraphanin has also been found to be soluble in The Cdrabawas harvested at maturity near Vernon, water and ethanol as it is a polar molecule.8 Broc- British Columbia, Canada in late April and early May coli sprouts are considered to be a substantial food of 2001. It was partially air-dried and then transported source of glucoraphanin, however this material is by bus to Saskatoon, Saskatchewan, Canada, where not currently consumed in any significant quantity the ambient drying was completed. by mankind. It would be beneficial if glucoraphanin could be extracted and purified and used as an additive Chemicals and enzymes to more commonly-consumed food materials. Numer- Reverse osmosis water that was also filtered using a ous plants of the Brassicaceae, including several weeds, Purelab Plus UV/UF US Filter (Lowell, MA, USA) contain significant amounts of glucoraphanin and pro- was used in all experimental work. Ethanol (95%) was vide new sources of the compound. These include obtained from Commercial Alcohols Inc (Brampton, plants of the Lepidium, Diplotaxis, and Eruca as well as Ontario). Pyridine, glacial acetic acid, hydrochloric 1 the model species Arabidopsis thaliana. acid, and sodium hydroxide were purchased from The noxious weed Cardaria draba (whitetop hoary BDH Inc (Toronto, Ontario). The acetonitrile (HPLC cress) was selected in this work as being the source grade) was from Merck (Darmstadt, Germany) 9 of glucoraphanin. McInnis indicated that the total and the compressed gases from Praxair Canada glucosinolate content in Cdrabaranges from 12 to Inc (Mississauga, Ontario). All other compounds −1 36 mg g . Cdrabacontains only two glucosinolates in including standards and enzymes were obtained significant amounts, glucoraphanin and glucosinalbin, from Sigma-Aldrich Chemicals (Oakville, Ontario). and therefore purification problems are minimized Glucoraphanin is not available commercially and an 1 compared with other plant sources of glucoraphanin. authentic standard had to be obtained by extraction The separation of individual glucosinolates is difficult and purification from plant tissues, similar to the because they are highly charged, and separation method used by Prestera et al.10 strategies often depend on small differences in the non-polar side chains.10 Apparatus and procedures Published work on glucosinolate removal from Glass, baffled extraction vessels capable of holding plants has focused on using batch liquid solvent 0.110 dm3 of solution were used to perform the extraction. The majority of these have been either series of optimization experiments (Fig 1). Starburst on a small scale for quantification purposes or aimed magnetic stirrers of diameter 0.60 of vessel diameter at removal from food and feed products without were used for stirring, and rubber stoppers were concern for glucosinolate degradation.11– 17 Faudet used to prevent solvent loss due to evaporation. et al18 studied the effects of several parameters on A nine-position Barnstead/Thermolyme PMC 730 solid–liquid extraction of rapeseed meal. In a single Series DataPlate Digital Hot Plate/Stirrer with probe contact batch extraction using water at 18 ◦C, and a (Dubaque, IA, USA) for temperature control was used solids loading of 167 g dm−3, equilibrium was achieved to perform simultaneous extraction runs. within 15 mins. They observed that the glucosinolate Dried Cdrabaleaves were ground for 1 min in extraction yield decreased with increasing ethanol 50 g allotments using a blender (BlendMaster 7, fraction in the aqueous solvent. Similar results were Proctor Silex, Washington, NC, USA). The contents obtained using methanol in place of ethanol, however methanol is toxic and is not a suitable solvent for nutraceutical products. A standard solvent extraction procedure for glucosi- nolates was performed in this study which consisted of two 5-min extractions in boiling 80% ethanol aqueous solutions. These two extractions are separated by a homogenization step. This standard protocol is sim- ilar to those reported by others.16,19– 22 Many of the results reported in this study are compared with the amounts of glucoraphanin and glucosinalbin recov- ered from the same plant material using this standard procedure. The objective of this study was to optimize liquid solvent leaching for maximum glucoraphanin recovery in a single batch solid–liquid contact by examining Figure 1. Schematic of extraction vessel for batch experiments.

986 J Chem Technol Biotechnol 80:985–991 (2005) Glucoraphanin extraction from Cardaria draba: Part 1 of the blender were then shaken briefly to mix, Glucosinolate quantification and the blending and mixing repeated three times. The solvent was evaporated using nitrogen from The particle size distribution for the final ground 2.0 × 10−3 dm3 of each liquid extract sample using plant material was determined using a laser particle a Reacti-Therm Heating Module (Pierce, Rockford, size analyzer (Mastersizer X, LongBench Model, IL, USA, Model 18 870) with Reacti-Vap evaporating Malvern Instruments Inc, Worcestershine, UK). This unit (Pierce, Model 18 780). The solid residue was × −3 3 distribution was checked periodically to ensure it was dissolved in 2.0 10 dm of water, and proteins × −3 3 consistent. The bulk density was estimated from the were precipitated out by adding 0.5 10 dm of volume occupied by a known mass of plant material. 0.5 M lead barium acetate and vortexing to mix. Samples were filtered using 1.0 × 10−3 dm3 of Celite Samples of plant material were added to the pre- (170 g dm−3 diatomaceous earth), vortexing to mix, heated stirred solvent at the selected solids loading letting stand for 1 min, and centrifuging at 3000 g such that the total volume in the extraction vessel for 3 min to separate. The glucosinolates were was 0 11 dm3. Once the contact time elapsed, the . bound using DEAE–Sephadex (10 g in 0.100 dm3, extract solution was separated from the depleted 0.5 M pyridine acetate buffer, filtered, washed, and plant material by vacuum filtration and stored frozen. suspended in 0.02 M pyridine acetate) on 10.0 × Samples of the same untreated plant material used 10−3 dm3 Poly-Prep chromatography columns (Bio- in each set of experiments were collected so that Rad Labs, California, USA) and rinsing with 0.02 M glucosinolates could be quantified using the standard pyridine acetate followed by water. Desulfo analogs extraction method in order to standardize the results. of the glucosinolates were prepared by allowing aryl The pH was altered using either hydrochloric acid or sulfatase (EC 3.1.6.1) to react on the column for sodium hydroxide depending on the desired value. 24 hs. The desulfoglucosinolates were eluted using The extraction variables to be optimized were 2.0 × 10−3 dm3 of Millipore water and the sulfatase solvent composition (ethanol %, v/v), solids loading, was deactivated by boiling for 3 mins. Samples were temperature, pH, and time. A modified single- filtered using a 0.2 µm nylon Acrodisc filter (Gelman factor method for the optimization study using Sciences, Ann Arbor, Michigan, USA) and stored successive substitution to determine search locations frozen. All samples were prepared for analysis in was employed. The initial conditions were estimated duplicate. from literature or experimental experience and each HPLC analysis was performed using a Hewlett Packard 1100 series liquid chromatograph and diode factor, normally beginning with the most important array detector (Waldbron, Germany) employing a (assumed), is varied over several levels singly while the mobile phase program of 2% phosphoric acid other variables are held constant. The maximal level for 10 mins, adding a linear gradient from 0 of each factor is then updated as results are obtained. to 12% acetonitrile for 30 mins, and holding at This process is repeated until either a satisfactory result 12% acetonitrile for 25 mins. The flow rate was is obtained or little change is observed. The selection 1.0 × 10−3 dm3 min−1 at 25 ◦C using a Phenomex of this type of experimental design is supported by Hypersil C18 ODS column (250 × 4.6mm)with5µm 23 24 previous researchers. , A constant stirring speed Hypersil guard column. UV detection was at 226 nm. of 500 rpm was used that provided suspension of O-nitrophenyl-α-D-galactopyranoside (a compound the particles at the greatest solids loading used in not naturally present in Cdraba) was added to the this study. The reproducibility of the results was samples as internal standard to validate the operation checked using six randomized runs performed at the of the HPLC. same extraction conditions. The uncertainty in the results was determined using a confidence interval. For 90% confidence, the uncertainty in the data is RESULTS AND DISCUSSION ±10% for glucoraphanin and ±6% for glucosinalbin Plant material (of the values measured) and these are the error values The dried Cdrabaparticles used in this study had presented for the extraction data. a moisture content of 9% on a dry basis, Sauter

Gls

1000 Grp 24.341

750 Sin 18.511

mAU 500 250 17.272 0 0 10 20 30

Figure 2. HPLC chromatogram (226 nm) of the glucosinolate profile of Cdrabaextracts. Glucoraphanin (Grp), Glucosinalbin (Gls), and Sinigrin (Sin) are identified.

J Chem Technol Biotechnol 80:985–991 (2005) 987 EE Powell et al mean particle size of 340 µm and bulk density of time used by Faudet et al18 who found that 15 min − 400 g dm 3. A sample HPLC chromatogram showing was sufficient to achieve equilibrium in a single batch glucosinolate peaks observed in Cdrabaextracts is contact between water (18 ◦C) and rapeseed meal shown in Fig 2. The HPLC analysis confirms that the (167 g dm−3 solids loading). only major glucosinolates present are glucoraphanin and glucosinalbin. This glucosinolate profile of the C Temperature effects draba extracts is consistent with the findings of Fahey The glucoraphanin yield increased at higher temper- 1 et al. atures, as shown in Fig 4. The values obtained at Using the standard extraction method, the gluco- temperatures of 60 ◦C and above are significantly dif- raphanin content of the Cdrabaleavesusedinthis ferent (two-tailed t-test, = 0 10) from those obtained −1 α . study was measured at 1% (or 10 mg g )onadry below that temperature. The glucosinalbin yield was basis. The glucoraphanin content of the Cdrabaused not significantly affected by temperature. Faudet 9 is particular to the specific harvest. The extract solu- et al.18 found that there was no significant yield ben- tion contains components other than glucosinolates efit to extracting above room temperature for total and the solvent. The clear, bright green color of glucosinolates. In this series of tests, there was no sta- the higher ethanolic extracts indicates the presence tistical difference in mass extracted between 60, 70, 25 of chlorophyll. The low alcohol and water extracts and 80 ◦C. Operating at 80 ◦C was difficult due to the were a clear, brownish green color. Quantification of high volatility of ethanol and preliminary experiments these components was not undertaken, however the carried out at pH 7.0 (Fig 3) had shown a higher glu- possibility of toxicity was investigated through the coraphanin recovery at 70 ◦C compared with 80 ◦C. hepatoma cell toxicity tests discussed in a subsequent On the other hand, high temperatures typically cause 26 paper. higher extraction rates. Two reasons for this are that Preliminary experimental tests at initial conditions ◦ both solubility and the mass transfer rate increase with common for plant extraction (80% ethanol at 80 C temperature. A temperature-induced increase in mass and pH 7) indicated that employing the well-mixed, transfer rates will cause equilibrium to be reached more baffled vessel technology yielded an increase in quickly. For these reasons, 70 ◦C was selected as the glucoraphanin extraction to twice that recovered using optimum extraction temperature for glucoraphanin the standard method for plant extraction. Figure 3 recovery from Cdraba. shows these recovery results which are normalized by taking the ratio of the mass of glucoraphanin extracted in the baffled vessel to that extracted using Solvent composition effects the standard protocol. These tests also demonstrated Varying the ethanol content of the extraction solvent that 500 rpm stirring speed and 20 min extraction had a significant effect on the yield of both major time were sufficient to reach equilibrium conditions glucosinolates. However, Fig 5 demonstrates that between the Cdrabaparticles and the extracting above 10% ethanol, no significant change (two- = solvent, since at the 90% confidence limit there were tailed t-test, α 0.10) in glucosinalbin recovery was no changes in the masses of glucoraphanin collected observed. The glucoraphanin yields at both 15% at times over 20 min. This is in line with the extraction and 20% ethanol are significantly higher (two-tailed t-test, α = 0.10) than at other solvent compositions. The maximum yield at 20% was observed at more

Figure 3. Glucoraphanin recovered in preliminary experiments (80% aqueous ethanol, pH 7, solids loading at 50 g dm−3. The ordinate is normalized by taking the ratio of the mass of glucoraphanin extracted Figure 4. Effect of temperature on glucoraphanin extraction (80% in the baffled vessel to that recovered using the standard protocol). aqueous ethanol, pH 6.5, solids loading at 50 g dm−3).

988 J Chem Technol Biotechnol 80:985–991 (2005) Glucoraphanin extraction from Cardaria draba: Part 1

Figure 5. Effect of solvent composition on glucosinolate extraction Figure 7. Effect of solids loading on glucosinolate extraction (20% − (70 ◦C, pH 6.5, solids loading at 50 g dm 3). aqueous ethanol, 70 ◦C, pH 3).

collected at pH 9.8 and 10.2. The glucoraphanin yield is significantly greater at pH 3 than at pH 10 (two-tailed t-test, α = 0.10). Once the optimal pH was found, the pH of the extract solution post- leaching was measured. For extraction at the optimal conditions (20% aqueous ethanol, 70 ◦C and pH 3, with solids loading at 50 g dm−3, stirred at 500 rpm, and extracted over 20 mins), the post-extraction pH was 5.4. The plant solids and extracted compounds possess a significant ability to neutralize the acidic solution. Finnigan and Lewis20 used acidic aqueous alcohol solvents at pH 4.5 in their glucosinolate optimization experiments. They followed this extraction with a second contact with water at pH 11.0 for additional glucosinolate leaching. There are multiple references in the literature to using an extraction solvent at basic pH.14,15,27 Figure 6. Effect of pH on extraction of glucosinolates (20% aqueous ethanol, 70 ◦C, with solids loading at 50 g dm−3). Solids loading effects The effects of solid loadings between the limits data points and hence was chosen as the optimum of 50 and 150 g dm−3 are shown on Fig 7. Above ethanol concentration for further extraction studies. 150 g dm−3, the solution was too thick to mix with any No reference was found in the literature to using an degree of effectiveness within the limitations of the aqueous 20% ethanol solvent to leach glucoraphanin extraction apparatus. Figure 7 provides not only the from plant material. The most common ethanol normalized but also the absolute recoveries of gluco- concentration used to extract for total glucosinolates raphanin. As expected, the yield of glucoraphanin 20 is 80%. Finnigan and Lewis found that extraction of and glucosinalbin decreased as the solids loading rapeseed with up to 70% aqueous alcohol in a single increased. With a higher solid to liquid ratio in the batch process resulted in glucosinolate recoveries extraction vessel, it is more difficult to leach the ranging from 40 to 60%. Decrease of solvent polarity greater mass of solute out of the solid particles. As thereafter decreased the yield. well, even with excess time for extraction, the solute concentration cannot surpass the upper equilibrium pH effects limit of the solvent. At the conditions used in this The effects of the initial solution pH on the extraction investigation, the maximum amount of glucoraphanin process are shown in Fig 6. A dual peak effect for that was extracted in a single batch contact was close glucoraphanin was observed at very disparate values; to 30 mg of glucoraphanin per gram of dried Cdraba. pH 3 and pH 10. The data from runs at pH 2.5 to Compared with the standard extraction conditions, pH 3.2 were accumulated and compared with the data this was observed to represent a three times greater

J Chem Technol Biotechnol 80:985–991 (2005) 989 EE Powell et al recovery of glucoraphanin from the Cdrabaleaves at fitting the value of the diffusion coefficient, D.It the same solids loading. was found that the best value of D was equal to 1 × 10−10 m2 s−1. For diffusion of a solute through Optimal extraction conditions a liquid occluded in a solid matrix, diffusivities − − − − The optimum conditions determined to maximize range between 10 8 m2 s 1 and 10 12 m2 s 1,witha − − glucoraphanin yield in a single batch extraction are characteristic value of 10 10 m2 s 1.29 The effective ethanol concentration = 20%, temperature = 70 ◦C, diffusivity measured in the present study is therefore pH = 3, solids loading = 50 g dm−3. The gluco- in agreement with the published values for diffusion raphanin extraction yield at these optimum conditions coefficients in a liquid occluded in a solid matrix, was found to be 30 mg g−1 of dried Cdraba, or 60% and this is therefore the mechanism by which of the total present in the solids. The ultimate recov- glucoraphanin moves through the plant material. ery was determined using repeated batch extractions of the plant material employing fresh solvent each time. The amount of glucoraphanin recovered in the CONCLUSIONS present, single-staged batch study improved on pre- The optimum liquid solvent extraction conditions for viously published work (using the standard extraction maximizing glucoraphanin recovery from Cdrabais a protocol described earlier) by nearly three-fold, how- 20% ethanol aqueous solvent at 70 ◦C, initial pH 3, ever the glucoraphanin yield was still only 60%. A and loaded at 50 g dm−3. The leaching is performed yield of 60% for a single contact extraction agrees with over 20 min using baffled vessels and stirring at the findings of Finnigan and Lewis using well-mixed 500 rpm. At the determined optimum conditions, 20 vessels for other plant chemicals. combined with baffled, well-mixed technology, a nearly three-fold increase in glucoraphanin recovery Extraction time course was achieved compared with the standard control With a set of extraction conditions to maximize recovery method. Up to 3% (dry basis) glucoraphanin glucoraphanin yield, the extraction time course was was obtained from Cdrabausing the optimum carefully measured. The results are presented in Fig 8. extraction conditions and described batch extraction The data (symbols) were fitted to the characteristic protocol. The glucoraphanin was found to diffuse diffusion equation for the removal of a solute from a through the leaf particles according to diffusion theory solid into a well-mixed solvent:28 in occluded liquid pores.   ∂C ∂2C 2 ∂C = D + (1) ∂t ∂r2 r ∂r ACKNOWLEDGEMENTS Crank provides an analytical solution to the above We owe special thanks to Richard Blondin, Arlene equation under the initial (C = CO) and boundary Drimmie, Ian McGregor, Vic Shewchuk, Connie conditions (dC/dr = 0atr = 0andC = KCL at r = a, Wong, and Mike Wormsbecker for their assistance. for all times) occurring in this experiment. The solid We also acknowledge the Natural Sciences and line on Fig 8 represents the best fit (least squares) Engineering Research Council of Canada and the solution of eqn (1) to the experimental data, after Saskatchewan Agriculture Development Fund for financial support of this study.

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