Optimization of conditions for supercritical fluid extraction of flavonoids from hops (Humulus lupulus L.)

Optimization of conditions for supercritical fluid extraction of flavonoids from hops ( Humulus lupulus L.) 1 first-author †E-mail:gqhe@zju.edu.cn; hpxiong@126.com †E-mail:gqhe@zju.edu.cn; hpxiong@126.com gqhe@zju.edu.cn 1 corresp ‡Corresponding Author ‡Corresponding Author †E-mail:gqhe@zju.edu.cn; hpxiong@126.com †E-mail:gqhe@zju.edu.cn; hpxiong@126.com gqhe@zju.edu.cn 1 1 1 2 Department of Food Science and Nutrition, Zhejiang University, Hangzhou 310029, China Department of Food Science and Nutrition, Zhejiang University, Hangzhou 310029, China Department of Food Science and Nutrition, University of Conakry PB 1147, Guinea Department of Food Science and Nutrition, University of Conakry PB 1147, Guinea Waste hops are good sources of flavonoids. Extraction of flavonoids from waste hops (SC-CO 2 extracted hops) using supercritical fluids technology was investigated. Various temperatures, pressures and concentrations of ethanol (modifier) and the ratio ( w / w ) of solvent to material were tested in this study. The results of single factor and orthogonal experiments showed that at 50 °C, 25 MPa, the ratio of solvent to material (50%), ethanol concentration (80%) resulted in maximum extraction yield flavonoids (7.8 mg/g). HPLC-MS analysis of the extracts indicated that flavonoids obtained were xanthohumol, the principal prenylflavonoid in hops. Optimization Supercritical fluid extraction Flavonoids Xanthohumol Hops ( Humulus lupulus L.) INTRODUCTION 1 The hop plant ( Humulus lupulus L., Cannabinaceae) is a dioecious twining perennial widely cultivated throughout the temperate zones of the world. The inflorescences (hops or hop cones) are used in breweries to give beer its characteristics such as flavor and aroma. In this respect, the bitter acids and essential oils have received much attention. Carbon dioxide (CO 2 ) is currently the most accepted solvent for the manufacture of hops extracts by breweries. Its extracts contain nearly all essential oils in hops, a high ratio of α-acids (humulones) to β-acids (less bitter lupulones), a small amount of hard resins, and minor traces of triglycerides, waxes, chlorophylls and inorganic salts (Laws et al., 1977 1977 ). These features distinguish CO 2 extracts from those made with conventional organic solvents, and consequently enable production of beers with a better balance of hops aroma and bitter flavor. Moreover, CO 2 extracts lack traces of unpleasant solvents. Therefore, supercritical CO 2 (SC-CO 2 ) extraction has become an industrial process for the production of brewery ingredients. Nowadays, supercritical CO 2 extraction of hops exists in Australia, Germany, UK, USA and China (Gardner, 1993 1993 ; Palmer and Ting, 1995 1995 ). However, SC-CO 2 extraction has its limitation. For example, polar compounds such as flavonoids cannot be extracted by SC-CO 2 alone (Anna et al., 2004 2004 ; Catchpole et al., 2004 2004 ; de Maria et al., 1997 1997 ). The flavonoids remain in the waste product of the hops processing industry. Hops are very rich sources of prenylflavonoids, which are secreted along with bitter acids and essential oils by the lupulin glands of the inflorescences (Stevens et al., 1998 1998 ). Xanthohumol is structurally a simple prenylated chalcone that exists only in the hop plant, where it is the main prenylflavonoid of the hop cones (Stevens and Page, 2004 2004 ). Xanthohumol and other prenylated chalcones are now attracting greater attention in the medical field. To date, some prenylflavonoids examined in vitro present many biological activities: inhibition of the growth of breast cancer (MCF-7) cells in a dose-dependent manner (Miranda et al., 1999 1999 ); inhibition of the cytochrome P450-mediated activation of procarcinogens (Henderson et al., 1998 1998 ); inducing the activity of the carcinogen-detoxifying enzyme, quinone reductase (Miranda et al., 2000 2000 ). Other biological activities include inhibition of bone resorption, inhibition of diacylglycerol acyltransferase and antimicrobial activities (Tobe et al., 1997 1997 ; Tabata et al., 1997 1997 ; Mizobuchi and Sato, 1984 1984 ). Furthermore, the 8-prenylnaringenin, which is also present in hops, has been recognized as the most potent phytoestrogen isolated to date (Milligan et al., 2000 2000 ). This new and exciting biological activity may lead to biomedical application of xanthohumol and 8-prenylnaringenin in the future. Considering the continuous decrease of hops market prices, hops growers are now very interested in alternative applications (Stevens and Page, 2004 2004 ). The comprehensive utilization of hops, especially the waste hops (SC-CO 2 extracted hops) are of utmost importance. The extraction and separation of flavonoids from waste hops with organic solvents have been conducted, but the extraction using supercritical fluid (with ethanol added as modifier) has not been investigated so far. The purpose of the present study was to screen out the optimum conditions of supercritical fluid extraction (SFE) of flavonoids from waste hops. MATERIALS AND METHODS 1 Plant material 2 Commercial pellets of hops (Qingdao big flower, produced in Nov. 2003) were purchased from Xinjiang (China). Samples were pulverized in a high-speed mixer-grinder and sieved by 40-mesh screen ( Dp ≤0.42 mm). Sieved powders were first extracted by supercritical carbon dioxide at 200 bar, 40 °C for 3 h (Jose et al., 2003 2003 ). The remainder (SC-CO 2 extracted hops) was kept in sealed plastic bags in a refrigerator (4 °C) until use for extraction of flavonoids by supercritical fluid. Apparatus for supercritical fluid extraction 2 The system consists of solvent and feed pumps, refrigeration module, 5 L/50 MPa and 1 L/50 MPa extraction vessels, and 2 L/30 MPa and 1 L/30 MPa absorbent vessels. Light-phase fluid (carbon dioxide) was supplied from the CO 2 cylinder by a high-pressure metering pump. Heavy-phase fluid (aqueous ethanol) was supplied to the system by means of a duplex high-pressure pump. CO 2 flow was controlled by pump displacement and was monitored with high-pressure mass-flow meter. Operating temperature was regulated in the extractor and separators by means of three thermo-static baths. A series of valves regulated the pressure in the extractor and separators. Assays 2 Flavonoids content was estimated with Zhuang and Yu ( 1992 1992 ) method. The constituents of flavonoids were determined by HPLC-MS methods. HPLC separations were achieved on a Zorbax Eclipse RP C 18 column (4.6 mm×150 mm, 5 μm) at 1 ml/min using a linear solvent gradient from 40% to 80% B (acetonitrile) in A (1% aqueous acetic acid) over 40 min, followed by 80% B for 20 min. Detection was routinely accomplished by monitoring the absorbance signals at 370 nm (Stevens et al., 1997 1997 ). For the mass spectrometry, Agilent 1100 LC/MSD SL mass spectrometer was operated using atmospheric pressure chemical ionization (APCI) source in the positive ion mode. Mass spectrometry conditions were as follows: drying gas temperature 350 °C, drying gas flow 13.0 L/min, nebulizer pressure 60 psig, vaporizer temperature 325 °C, capillary voltage 4 000 V, corona current 4 μA, mass range 200~450, fragmentor 100 V. Experimental design and statistical analysis 2 1. Single factor experiments design 3 Four factors were tested respectively for the relation between their variation and the yields of flavonoids. The extraction time, temperature, pressure, ethanol concentration and the ratio of solvent to material (the amount of modifier) were fixed at 90 min, 50 °C, 200 bar, 70%, 40% (with each of them as a variation factor). 2. Orthogonal experiment design 3 The following four variable factors: temperature, pressure, modifier (ethanol) concentration and the amount of modifier were used to optimize the extraction process according to the design in Table 1 1 . Factor/level 1 2 3 A: extraction temperature (°C) 40 50 60 B: extraction pressure (MPa) 20 25 30 C: the amount of modifier (%) 30 40 50 D: ethanol concentration (%) 70 80 90 3. Statistical analysis 3 Every experiment was replicated three times. Data of interest were analyzed with LSD (least significant data test) method. DPS (data processing software) was used to design and process experiment in this study. RESULTS AND DISCUSSION 1 Effect of the amount of ethanol added on the yield of flavonoids 2 Considering one of the aims of this work is to propose a suitable quality control method for extracting flavonoids from hops in industrial processes, ethanol was selected as a modifier because it is environmentally benign and relatively safe to human health. The amounts of ethanol added were 30%, 40%, 50% and 60% of the total sample ( w / w ), respectively. Fig. 1 1 shows an increase of ethanol percentage up to 50% resulted in an increase of flavonoids recovery. This was consistent with the results of Lin et al.( 1999 1999 ) that under certain temperature and pressure, the higher the percentage of modifiers, the higher was the extraction yield achieved. However, ethanol above 50% exhibited a tremendous drop in flavonoids yield. It should be noted that the addition of large amounts of modifier would considerably change the critical parameters of the mixture and that the fluids were no longer supercritical above this value (Tong and Imagawa, 1995 1995 ; van der Velde et al., 1994 1994 ). Therefore, appropriate amount of ethanol in supercritical fluid is very crucial for flavonoids extraction. Effect of ethanol concentration on the yield of flavonoids 2 The results of ethanol concentration on the recovery of the flavonoids are shown in Fig. 2 2 . The yield of flavonoids increases with an increase in ethanol concentration until 80%. Ethanol concentration beyond 80% resulted in a reduction of flavonoids recovery. Ethanol added to the supercritical CO 2 increases the polarity of the fluid. The modifier exerts its effect mainly in two basic ways: by interacting with the analyte complex to promote rapid desorption into the supercritical fluid, and by enhancing the solubility properties of supercritical CO 2 (Luque de Castro and Tena, 1996 1996 ). Various concentration of ethanol used exhibited different effect in changing the fluid polarity and thus had diverse effect on the solubility enhancement of the flavonoids. The optimal extraction yield may be fulfilled when the polarity of the fluid and its flavonoids are coincident. In this study, the results indicated that the optimal ethanol concentration for extraction flavonoids of hops was 80%. Effect of pressure on the yield of flavonoids 2 Fig. 3 3 shows that the flavonoids yield was pressure dependent because higher pressure in all cases favors higher recovery of flavonoids. Higher pressure can increase the bulk density of the fluid mixture that would contribute to solubility enhancement. However, as pressures increase further, the fluid is less compressible, the increase in bulk density is not expected to be very significant (Li et al., 2003 2003 ). Based on the above findings and the feasibility in industrial processes, pressure over 35 MPa was not considered in this study. Effect of temperature on the yield of flavonoids 2 Fig. 4 4 shows that temperature of 40 °C to 50 °C resulted increased yield. The extraction yield had decreasing trend when temperature increased from 50 °C to 60 °C. Further increase from 60 °C to 70 °C led again increased yield but the recovery rate was lower than that at 50 °C. Temperature’s effect on extraction is dual. On one hand, higher temperature can accelerate fluid flow and thus increase the solute of compounds; on the other hand, higher temperature can decrease fluid density and thus reduce extraction efficiency. The overall extraction effect of supercritical fluids usually follows the competition between the increasing in solute of compounds and the reduction in SC-CO 2 density due to the rise in temperature (Chiu et al., 2002 2002 ). Orthogonal experiments 2 The optimum conditions for the extraction were obtained by using orthogonal design (L 9 3 4 ) based on single factor experiments. Table 1 1 lists the coded levels of the orthogonal test factors. Table 2 2 shows factors at different levels in nine experiments conducted and the statistical analysis results. The order of the effect of factors on flavonoids extraction was found to be: B > A > C > D. The optimum extraction conditions obtained from the statistical analysis were A 2 B 2 C 3 D 2 . It means that 50 °C, 25 MPa, 50% (amount of modifier), 80% (ethanol concentration) were the optimum conditions for flavonoids recovery by supercritical fluid extraction. These conditions were tested later to ascertain the dependability of our results; the recovery of flavonoids was 7.8 mg/g. So the hypothesis of the orthogonal experiment was valid. Run A B C D Content of flavonoids (mg/g) 1 1 1 1 1 3.74 2 1 2 2 2 6.19 3 1 3 3 3 6.42 4 2 1 2 3 4.50 5 2 2 3 1 6.67 6 2 3 1 2 5.82 7 3 1 3 2 4.82 8 3 2 1 3 4.48 9 3 3 2 1 3.76 K 1 16.35 13.05 14.04 14.17 K 2 16.99 17.34 14.45 16.83 K 3 13.06 16.01 17.90 15.40 R 1.18 1.28 1.16 0.80 HPLC-MS results 2 The conditions of HPLC-MS were described above. A total ion chromatogram of SFE extract is shown in Fig. 5 5 . Since the standards of flavonoids of hops were not available, the results compared with those reported in Stevens et al.( 1997 1997 ; 1999 1999 ) based on polarity (retention time), m/z, and relative abundance. Three compounds were proposed. Some flavonoids such as 8-prenylnaringenin, 6-prenylnaringenin, desmethylxanthohumol and so on could not be identified in this study; while other compounds such as peak 4 and peak 5 have not been reported up to now (Table 3 3 ). Different varieties of hops and extraction methods could have led to the diversity between our study and other related research (Stevens et al., 1997 1997 ; 1999 1999 ). Those unknown compounds will be investigated and identified in the future study. RT : retention time Peak number RT (min) * * [M+H] + (m/z) Proposed compound 1 5.864 355 Isoxanthohumol 2 19.239 355 Xanthohumol 3 26.389 353 Dehydrocycloxanthohumol 4 29.902 369 Unknown 5 33.445 385 Unknown CONCLUSION 1 The results presented in this work indicated that SFE was feasible for extraction of flavonoids from waste hops, especially for the extraction of xanthohumol, which was reported to have multiple biological activities. Extraction conditions was optimized to find that extraction temperature 50 °C, 25 MPa, the ratio of solvent to material (50%), ethanol concentration 80% were the optimal conditions. HPLC-MS analysis results revealed three purported to exist compounds and two unknown compounds. Considering other parameters such as particle size of samples, fluid flow rate of fluid, etc., simultaneously may require further optimization. More research on flavonoids biological activity should be done in the future research. References M Anna , K Marina , K Mihkel , O Anne . Identification and characterization of supercritical fluid extracts from herbs . C R Chimie , 2004 . 7 629 - 633 . . Identification and characterization of supercritical fluid extracts from herbs OJ Catchpole , JB Grey , KA Mitchell , JS Lan . Supercritical antisolvent fractionation of propolis tincture . Journal of Supercritical Fluids , 2004 . 29 97 - 106 . . Supercritical antisolvent fractionation of propolis tincture KL Chiu , YC Cheng , JH Chen , CJ Chang , PW Yang . Supercritical fluids extraction of Ginkgo ginkgolides and flavonoids . Journal of Supercritical Fluids , 2002 . 24 77 - 87 . . Supercritical fluids extraction of Ginkgo ginkgolides and flavonoids LLM de Maria , HYV Janete , ML Fernando . Supercritical fluid extraction of glycosylated flavonoids from Passiflora leaves . Phytochemical Analysis , 1997 . 8 257 - 260 . . Supercritical fluid extraction of glycosylated flavonoids from Passiflora leaves DS Gardner . MB King , TR Bott . Commercial Scale Extraction of Alpha-Acids and Hop Oils with Compressed CO 2 . Extraction of Natural Products Using Near-Critical Solvents , 1993 . : London, UK Blackie Academic and Professional , 84 - 100 . . Commercial Scale Extraction of Alpha-Acids and Hop Oils with Compressed CO2 MC Henderson , CL Miranda , JF Stevens , ML Deinzer , DR Buhler . In vitro inhibition of carcinogen metabolism by flavonoids from hops ( Humulus lupulus ) in various species . The Toxicologist , 1998 . 42 185 . In vitro inhibition of carcinogen metabolism by flavonoids from hops (Humulus lupulus) in various species M Jose , R Oscar , T Osvaldo , MT Palma . Supercritical CO 2 extraction of Chilean hop ( Humulus lupulus ) ecotypes . Journal of the Science of Food and Agriculture , 2003 . 83 1349 - 1356 . . Supercritical CO2 extraction of Chilean hop (Humulus lupulus) ecotypes DR Laws , NA Bath , JA Pickett , CS Ennis , AG Wheldon . Preparation of hop extracts without using organic solvents . Journal of the Institute of Brewing , 1977 . 83 39 - 40 . . Preparation of hop extracts without using organic solvents QS Li , ZT Zhang , CL Zhong , YC Liu , QR Zhou . Solubility of solid solutes in supercritical carbon dioxide with and without cosolvents . Fluid Phase Equilibria , 2003 . 207 183 - 192 . . Solubility of solid solutes in supercritical carbon dioxide with and without cosolvents MC Lin , MJ Tsai , KC Wen . Supercritical fluid extraction of flavonoids from Scutellariae Radix . Journal of Chromatography A , 1999 . 830 387 - 395 . . Supercritical fluid extraction of flavonoids from Scutellariae Radix MD Luque de Castro , MT Tena . Strategies for supercritical fluid extraction of polar and ionic compounds . Trends in Analytical Chemistry , 1996 . 15 32 - 37 . . Strategies for supercritical fluid extraction of polar and ionic compounds SR Milligan , JC Kalita , V Pocock , V Kauter , JF Stevens , ML Deinzer , H Rong , D Keukeleire . The endocrine activities of 8-prenylnaringenin and related hop ( Humulus lupulus L.) flavonoids . Journal of Clinical Endocrinology and Metabolism , 2000 . 85 4912 - 4915 . . The endocrine activities of 8-prenylnaringenin and related hop (Humulus lupulus L.) flavonoids CL Miranda , JF Stevens , A Helmrich , MC Henderson , RJ Rodriguez , ML Deinzer , DW Barnes , DR Buhler . Antiproliferative and cytotoxic effects of prenylated flavonoids from hops ( Humulus lupulus ) in human cancer cell lines . Food and Chemical Toxicology , 1999 . 37 271 - 285 . . Antiproliferative and cytotoxic effects of prenylated flavonoids from hops (Humulus lupulus) in human cancer cell lines CL Miranda , L Aponso , JF Stevens , ML Deinzer , DR Buhler . Prenylated chalcones and flavanones as inducers of quinone reductase in mouse hepatoma (Hepa lclc7) cells . Cancer Letters , 2000 . 149 21 - 39 . . Prenylated chalcones and flavanones as inducers of quinone reductase in mouse hepatoma (Hepa lclc7) cells S Mizobuchi , Y Sato . A new flavanone with antifungal activity isolated from hops . Agriculture Biology Chemistry , 1984 . 48 2771 - 2775 . . A new flavanone with antifungal activity isolated from hops MV Palmer , SST Ting . Applications for supercritical fluid technology in food processing . Food Chemistry , 1995 . 52 345 - 352 . . Applications for supercritical fluid technology in food processing JF Stevens , JE Page . Xanthohumol and related prenylflavonoids from hops and beer: to your good health . Phytochemistry , 2004 . 65 1317 - 1330 . . Xanthohumol and related prenylflavonoids from hops and beer: to your good health JF Stevens , I Monika , LH Victor , ML Deinzer . Prenylflavoniods from Humulus lupulus . Phytochemistry , 1997 . 44 1575 - 1585 . . Prenylflavoniods from Humulus lupulus JF Stevens , CL Miranda , DR Buhler , ML Deinzer . Chemistry and biology of hop flavonoids . Journal of the American Society of Brewing Chemists , 1998 . 56 136 - 145 . . Chemistry and biology of hop flavonoids JF Stevens , AW Taylor , ML Deinzer . Quantitative analysis of xanthohumol and related prenylflavonoids in hops and beer by liquid chromatography-tandem mass spectrometry . Journal of Chromatography A , 1999 . 832 97 - 107 . . Quantitative analysis of xanthohumol and related prenylflavonoids in hops and beer by liquid chromatography-tandem mass spectrometry N Tabata , M Ito , H Tomoda , S Omura . Xanthohumols, diacylycerol acyltransrerase inhibitors, from Humulus lupulus . Phytochemistry , 1997 . 46 683 - 687 . . Xanthohumols, diacylycerol acyltransrerase inhibitors, from Humulus lupulus H Tobe , Y Muraki , K Kitamura , O Komiyama , Y Sato , T Sugioka , HB Maruyama , E Matsuda , M Nagai . Bone resorption inhibitors from hop extract . Bioscience Biotechnology Biochemistry , 1997 . 61 158 - 159 . . Bone resorption inhibitors from hop extract P Tong , T Imagawa . Optimization of supercritical fluid extraction for polychlorinated biphenyls from sediments . Analytica Chimica Acta , 1995 . 310 93 - 100 . . Optimization of supercritical fluid extraction for polychlorinated biphenyls from sediments EG van der Velde , M Dietvorst , CP Swart , MR Pamlal , PR Kootstra . Optimization of supercritical fluid extraction of organochlorine pesticides from real soil samples . Journal of Chromatography A , 1994 . 683 167 - 174 . . Optimization of supercritical fluid extraction of organochlorine pesticides from real soil samples XP Zhuang , XY Yu . Determination of total flavonoids in the leaves of Ginkgo and studies on its extraction process . Chinese Traditional and Herbal Drugs , 1992 . 23 122 - 124 . (in Chinese) . Determination of total flavonoids in the leaves of Ginkgo and studies on its extraction process 10.1631/jzus.2005.B0999 Project (No. Y304203) supported by the Natural Science Foundation of Zhejiang Province, China 2005-03-01 2005-06-09 2005-09-28 2022-01-05 Copyright © 2005, Journal of Zhejiang University Science 2005 Journal of Zhejiang University-SCIENCE B (Biomedicine and Biotechnology) 10 6B