JOURNAL OF LABELLED COMPOUNDS AND RADIOPHARMACEUTICALS J Label Compd Radiopharm 2006; 49: 479–487. Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jlcr.1057 Research Article Synthesis of deuterated dihydrochalcones Daniel J. Comeskey1,*, Janine M. Cooney2 and Daryl D. Rowan1 1 The Horticulture and Food Research Institute of New Zealand Ltd, Tennent Drive, Private Bag 11030, Palmerston North, New Zealand 2 The Horticulture and Food Research Institute of New Zealand Ltd, East Street, Private Bag 3123, Hamilton, New Zealand Summary The dihydrochalcones phloretin and phloridzin are major phenolic constituents of apple fruit. Phloretin-d4, deuterated at both the a and b positions, was prepared by hydrogenolysis of naringenin and by deuterium exchange from unlabelled phloretin using Pd/C and sodium formate with methanol-d1 as the source of deuterium. Deuterated derivatives of the glycosides, phloridzin and naringin dihydrochalcone, were similarly prepared. Copyright # 2006 John Wiley & Sons, Ltd. Key Words: dihydrochalcone; phloretin; phloridzin; naringin dihydrochalcone; deuterium; synthesis Introduction Dihydrochalcones are a class of ‘minor flavanoids’ that widely occur in nature both as the glycosides and free aglycones.1 New dihydrochalcones, variously methoxylated2 and with chromyl,3 galloyl, caffeoyl and hexahydroxydiphenoyl ester4 and C-b-glucopyranosyl derivatives5 continue to be reported. The dihydrochalcones, phloretin (3-(4-hydroxyphenyl)-1-(2,4,6-trihydroxyphe- nyl)propan-1-one, Figure 1(a)) and phloridzin (1-[4,6-dihydroxy-2-O- (b-d-glucopyranosyl)phenyl]-3-(4-hydroxyphenyl)propan-1-one, Figure 1(b)) are found throughout the apple tree1 and are regarded as the characteristic phenolics of apple fruit and of apple products, with concentrations of phloridzin in fruit ranging from 0.1 to 190 mg/kg.6 Phloridzin has also been reported from various Malus, Prunus and Populus species1 and from straw- berry.7 Phloridzin is reportedly repellent to blackbirds, its consumption may *Correspondence to: D. J. Comeskey, The Horticulture and Food Research Institute of New Zealand Ltd, Private Bag 11030, Palmerston North, New Zealand. E-mail: dcomesky@hortresearch.co.nz Contract/grant sponsor: Foundation for Research Science and Technology; contract/grant number: COX0220 Copyright # 2006 John Wiley & Sons, Ltd. Received 11 December 2005 Revised 12 December 2005 Accepted 13 January 2006 480 D. J. COMESKEY ET AL. HO OH OH 5' 3 HO OH 4 OH 2 A α B O O O 3' HO β OH O HO OH OH (a) (b) Figure 1. Structures of phloretin (a) and phloridzin (b). The numbering scheme follows Bohm1 confer deterrence to insects feeding on apple1 and it has strong antioxidant activity.8 The biological activity of polyphenolic antioxidants and their possible role in promoting health, when consumed as part of a normal diet, is the subject of much recent research. Apples and apple products are the major source of polyphenolic dihydrochalcones in the human diet with 250 ml of apple juice or cider estimated to supply 1–5 mg of phloretin and a whole dessert apple (c. 100 g) supplying about 1 mg.6 Phloridzin competitively inhibits glucose uptake by the sodium glucose cotransporter 1(SGLT1) in the small intestine,9 has been classified as an anti-diabetic agent10,11 and continues to be used as a research tool in the study of diabetes.12 In rats, phloretin is excreted in the urine principally as phloretic acid, (4-hydroxyphenol)propr- ionic acid, and related metabolites presumably as a result of microbial metabolism in the gut.13 In plasma, phloridzin occurs largely as phloretin conjugates.14 To further understand the biological activity of this class of compounds, isotopically labelled derivatives would be useful. Methods for the synthesis of labelled phenolics generally involve hydrogen exchange under acidic,15 basic or supercritical16,17 conditions with a back exchange step sometimes being used to remove unwanted label from the most labile positions.18 Many complex phenolics do not survive these reaction conditions.15 Krishnamurty and Sathyanarayan19 reported the synthesis of dihydrochalcones from flavanones by catalytic hydrogenation using sodium formate and Pd/C. This method seemed applicable to the incorporation of an isotope label (deuterium or tritium) in the non-exchangeable b position of the dihydrochalcone skeleton. We report here the further development of this chemistry to synthesize deuterated dihydrochalcones both by hydrogenolysis from the corresponding flavanones and by deuterium exchange from dihydro- chalcone aglycones or glycosides using methanol-d1 as the deuterium source. Copyright # 2006 John Wiley & Sons, Ltd. J Label Compd Radiopharm 2006; 49: 479–487  SYNTHESIS OF DEUTERATED DIHYDROCHALCONES 481 Results and discussion Reaction of naringenin (40 ,5,7-trihydroxyflavan-4-one, 2) with sodium formate-d1 (DCOONa) and formic acid-d2 (DCOOD) in refluxing isopropanol gave phloretin 1a in 33% yield as reported19 but with little incorporation of deuterium (515% by MS) and with less than 5% deuteration a or b to the carbonyl group as measured by NMR. Most of the deuterium exchange had occurred on ring A at C30 and C50 presumably by deuterium exchange occurring under the highly basic conditions occurring at completion of the reaction. Repetition of the reaction in methanol-d1, and optimization of the reaction workup, gave deuterated phloretin 1a-d4 (Table 1) in 84% yield (99% pure by HPLC). FDMS gave a molecular ion cluster centred at m/z 278.1108 indicating incorporation of four deuterium atoms. Integration of residual signals in the 1H NMR at d 3.28 and 2.84 ppm showed 88 and 93% deuterium incorporation, respectively, at positions a and b to the carbonyl group. Some additional deuteration into ring A (25% distributed between H30 and H50 ) was also observed. Reaction in methanol-d1 using non-deuterated sodium formate and with omission of the formic acid20 similarly gave 1a-d4 with the same level of deuterium incorporation. Incorporation of four deuterium atoms into 1a-d4 indicated that more complex chemistry than simple hydrogen transfer19 had occurred. Deuterium exchange adjacent to the carbonyl group can be accounted for by the highly alkaline conditions (pH>12) present at the end of the reaction but the pres- ence of two deuterium atoms b to the carbonyl group suggested palladium benzylic catalysed exchange had also occurred. To test this, the reaction was repeated using phloretin 1a as the substrate. Reaction of phloretin 1a with sodium formate in refluxing methanol-d1 gave 1a-d4 in 84% yield (99% pure by HPLC) with 91% incorporation of deuterium a and b to the carbonyl group as judged by 1H NMR. Deuteration resulting from deuterium transfer from methanol-d1 implies formate19 is not the sole hydrogen donor in this reaction. Following Rajagopal and Spatola,20 an alternative scheme, involving addition of methanol-d1 to a reduced palladium species (Figure 2), is proposed to account for the extensive deuteration observed in the reaction. The deuterium atoms a to the carbonyl group are potentially exchangeable and the removal of the deuterium from this site by back exchange was briefly investigated. Reaction of 1a-d4 with 1.0 M sodium hydroxide in methanol for 24 h at room temperature reduced deuteration at the a position from 88 to 75%. Deuteration at the b position was unchanged. Under prolonged or harsher reaction conditions 1a-d4 showed decomposition. The deuteration a to the carbonyl group was surprisingly stable implying participation by palla- dium in the exchange reaction. Copyright # 2006 John Wiley & Sons, Ltd. J Label Compd Radiopharm 2006; 49: 479–487  Table 1. Percent deuteration as determined by 1H NMR of synthesized dihydrochalcones 482 Substrate Product % Deuteration OH HO OH OH HO O D D D 88(Ha), 93(Hb) OH O D OH O 1a-d4 2 HO OH OH HO OH OH D D D 91(Ha), 91(Hb) OH O OH O D Copyright # 2006 John Wiley & Sons, Ltd. 1a 1a-d4 HO OH OH HO OH OH D D D O O O O O O D HO HO 94(Hb) D. J. COMESKEY ET AL. HO OH HO OH OH OH 1b 1b-d4 OH HO OH OH O O D HO O O O OH OH D OH HO D O O OH D 96(Ha), 95(Hb) OH HO O D O HO O O OH OH OH OH J Label Compd Radiopharm 2006; 49: 479–487 3-d5 3  SYNTHESIS OF DEUTERATED DIHYDROCHALCONES 483 Pd/C HCOO- CO2 RH CO2 HCOO- PdH- H MeO MeO- - Pd Pd- H RD R MeO - H2 Pd Pd- D R R MeOD Figure 2. Mechanism proposed for the deuterium exchange of dihydrochalcones using Pd/C and sodium formate in methanol-d1 The generality of this deuterium exchange reaction was tested using the glycosides phloridzin 1b and naringin (40 ,5-dihydroxy-7-O-(a-l- rhamnopyranosyl(1 ! 2)-b-d-glucopyranosyl)flavan-4-one 3) (Table 1). Reac- tion of 1b with Pd/C and sodium formate in methanol-d1 gave deuterated phloridzin 1b-d4 in 29% isolated yield (95% pure by HPLC). FDMS gave the most abundant molecular ion at m/z 440 corresponding to the incorpo- ration of four deuterium atoms. A fragment ion at m/z 278 confirmed deuteration incorporation into the aglycone. 1H NMR analysis showed 94% deuterium incorporation at the b position; however, residual signals for the a protons were obscured by signals from H3 of glucose. Irradiation of the b protons at d 2.87 in a TOCSY experiment confirmed their attachment to a weak doublet centred at d 3.46. Integration of signal intensities also indicated some deuterium incorporation into the A ring (57 and 20% incorporation at H30 and H50 , respectively). Extensive deuteration of 1b-d4 at the a position was confirmed by LC-MS/MS. Thus negative ionization of 1b-d4 gave a pseudomolecular ion m/z 439 (M-H)
which was fragmented with an initial loss of glucose to give an aglycone ion (ms2) at m/z 277 (C15H9D4O
3 5 ). This daughter ion was in turn fragmented (ms ) to give two fragment ions derived from ring A at m/z 125 (C6H5O
3 ) and m/z 169 (C8H5O4D
2 ). In an attempt to increase the yield of phloridzin 1b-d4, shorter reaction times and more mild conditions were investigated. Comparable deuterium incorporation and recoveries were obtained after 30 min in refluxing metha- nol-d1. No deuterium exchange was observed after 4 h of reaction at room temperature. Copyright # 2006 John Wiley & Sons, Ltd. J Label Compd Radiopharm 2006; 49: 479–487 484 D. J. COMESKEY ET AL. Reaction of naringin 3 under the standard reaction conditions gave deute- rated naringin dihydrochalcone (1-[2,6-dihydroxy-4-O-(a-l-rhamnopyranosyl (1 ! 2)-b-d-glucopyranosyl)-3-2H1-phenyl]-3-(4-hydroxyphenyl)-[2,2,3,3]-2H4- propan-1-one 3-d5) in 27% yield (98% pure by HPLC). FDMS gave the molecular ion as the sodium adduct at m/z 610.2145 ((M+Na)+, C27H30D5O14Na) together with a less abundant ion cluster centred at m/z 587 (M+) and a major fragmentation ion centred at m/z 278.1099 (C15H10D4O5) confirming deuteration of the aglycone. LCMS analysis of 3- d5 showed a narrower distribution of deuterated species with a pseudomolec- ular ion cluster centred at m/z 587 (M-H)
, six mass units above that of non- deuterated naringin dihydrochalcone ((M-H)
, m/z 581). The reason for the difference in isotope distributions between these two MS methods is not known but the formation of both M+ and (M+H)+ ions during FDMS has been reported.21 Based on the LCMS results, negative ion LC-MS/MS of both 3-d5 and of non-deuterated naringin dihydrochalcone was used to search for any addi- tional sites of deuteration in 3-d5. Fragmentation of the deuterated pseudo- molecular ion m/z 587 (M-H)
gave prominent daughter ions at m/z 479 (loss of 108, C7H4D2O, ring B) and at m/z 466 (loss of 121, presumably a sugar fragment C4H7D1O4) ms3 fragmentation of the ion at m/z 466 containing five deuterium atoms gave fragment ions at m/z 357 (loss of 108, C7H4D2O), at m/z 278 (aglycone, C15H9D5O

5 ) and at m/z 277 (aglycone, C15H9D4O5 ) consistent with partial additional deuteration on ring A of the dihydrochalcone and complete deuteration at both the a and b positions. Detailed 1H NMR analysis of 3-d5 in d6-acetone (using COSY, TOCSY, HSQC and HMBC experiments and both deuterated and non-deuterated compounds to aid assignments) showed 96 and 95% deuteration at Ha and Hb, respectively, and also indicated extensive deuteration in ring A (81% on average at H30 and H50 ). No further deuteration was detected elsewhere in the molecule. The origens of the higher mass ions recorded by LCMS with 3-d5 remain unknown. Experimental General Reagents were obtained from the Aldrich Chemical Co. (Milwaukee, WI) and Kodak (naringin) and were used without further purification unless otherwise stated. 1H and 13C nuclear magnetic resonance (NMR) spectra were recorded on Bruker 400 and 500 NMR spectrometers. Chemical shifts (d) are in parts per million relative to acetone-d6 at 2.15 ppm for 1H and at 30.67 ppm for 13C. The following abbreviations are used: s, singlet; d, doublet; t, triplet; m, multiplet; br, broad. Field desorption mass spectra (FDMS) were recorded on Copyright # 2006 John Wiley & Sons, Ltd. J Label Compd Radiopharm 2006; 49: 479–487  SYNTHESIS OF DEUTERATED DIHYDROCHALCONES 485 a Waters GCT time of flight mass spectrometer equipped with a FD probe operating with an extraction voltage of 12 kV and ramping the emitter current from 0 to 70 mA over 8 min. Pentafluorochlorobenzene was used as the lock mass. LCMS-MS spectrometry was carried out using a LCQ Deca ion trap mass spectrometer fitted with an ESI interface (ThermoQuest, Finnigan, San Jose, CA, USA) and coupled to a SurveyorTM HPLC and PDA detector. Analysis was by direct infusion of the sample at 10 ml/min. Full-scan mass spectral data were acquired in the negative mode. MS/MS data were acquired by isolation and fragmentation of the M-1 parent ion to give ms2 data, fol- lowed by isolation and fragmentation of the most intense daughter ion to give ms3 spectra. Phloretin-d4 1a-d4 . Typically to a solution of phloretin 1a or naringenin 2 (50 mg) in methanol-d1 (3 ml) was added sodium formate (50 mg) and palla- dium on charcoal powder (50 mg). The reaction mixture was stirred at reflux for 4 h before being filtered through a plug of celite with methanol washings. The solution was acidified with 1 M HCl and the methanol removed in vacuo. The resulting aqueous solution was extracted with ethyl acetate (50 ml), and the resulting organic layer washed with water, saturated brine, and then dried over magnesium sulphate, filtered and the solvent removed in vacuo. The crude product was purified by column chromatography on silica, eluting with meth- anol/dichloromethane (6:94) to give 1a-d4 (42 mg, 84%) as a white solid. 1H NMR d 7.06 (2H, d, J=8.8 Hz, H2, H6), 6.72 (2H, d, J=8.8 H3, H5), 5.93 (1.75H, s, H30 , H50 ) 3.28 (0.24H, s, Ha), 2.84 (0.14H, s, Hb) ppm.22 13C NMR d 205.6, 165.4, 165.2, 156.3, 133.3, 130.1, 115.9, 105.1, 95.7 ppm. FDMS m/z 278.1108 (M+, C15H10D4O5 requires 278.1092, isotopic distribution d2:d3:d4:d5:d6=5:36:100:28:4). Back exchange of phloretin-d4. To a solution of 1a-d4 (30.0 mg, 0.11 mmol) in methanol (5 ml) at room temperature was added NaOH (200 mg, 5 mmol). After stirring for 24 h at room temperature, the reaction mixture was neu- tralized with 1 M HCl, the methanol removed in vacuo and the residue ex- tracted with ethyl acetate. The ethyl acetate phase was washed with water then brine, then dried over magnesium sulphate and the solvent removed in vacuo to give a white solid (20.9 mg, 70%). 1H NMR d 7.09 (2H, d, J=8.8 Hz, H2, H6), 6.74 (2H, d, J=8.8 Hz, H3, H5), 5.95 (1.86H, s, H30 , H50 ) 3.28 (0.50H, s, Ha), 2.85 (0.14H, s, Hb) ppm. FDMS m/z 277.1020 (M+, isotopic distribution d1:d2:d3:d4:d5=12:86:100:49:10). Deuterated glycosides 1b-d 4 , 3-d 5 . Typically to a solution of substrate (500 mg) in methanol-d1 (20 ml) was added sodium formate (500 mg) and palladium on charcoal powder (500 mg). The reaction mixture was stirred at reflux for Copyright # 2006 John Wiley & Sons, Ltd. J Label Compd Radiopharm 2006; 49: 479–487 486 D. J. COMESKEY ET AL. 30 min before being filtered through a plug of celite, the solution was acidified with 1 M HCl and the methanol removed in vacuo. The crude product was filtered through reverse-phase silica with water, followed by water/methanol (1:1). The water/methanol fraction was further purified by chromatography on silica eluting with methanol/dichloromethane (1:9). Removal of solvent in vacuo gave a glassy solid that was recrystallized from water to give the product as a white solid. Phloridzin 1b-d 4 (145 mg, 29%) 1H NMR d 7.13 (d, 2H, J=6.5 Hz, H2, H6), 6.75 (d, 2H, J=6.5 Hz, H3, H5), 6.29 (0.80H, br s, H50 ), 6.02 (0.43H, d, J=2.1 Hz, H30 ), 5.13 (1H, d, J=5.7 Hz, Glu-H1), 3.92 (1H, dd, J=2.5, 11.9 Hz, Glu-H6a), 3.74 (1H, dd, J=5.7, 11.9 Hz, Glu-H6b), 3.57 (3H, m, Glu- H2, Glu-H4, Glu-H5), 3.46 (1H, m, Glu-H3), 2.87 (0.13H, s) ppm. 13C NMR d 161.3, 155.4, 132.4, 129.3 (C2), 115.0 (C3), 105.4, 101.2, 97.2 (C30 ), 94.4 (C50 ), 77.4, 77.1, 73.5, 70.3, 61.7 ppm. FDMS m/z 440.1703 (M+, C21H20D4O10 re- quires 440.1621), 422.1554 (M+-H2O, C21H18D4O9 requires 422.1515), 278.1099 (C15H10D4O5 requires 278.1092); LCMS-MS m/z 439 (M-H
, iso- topic distribution d2:d3:d4:d5=9:36:100:19); 277 (ms2), 169, 125 (ms3). Naringin dihydrochalcone 3-d 5 . (137 mg, 27%). 1H NMR d 7.09 (2H, d, J=8.5 Hz, H2, H6), 6.75 (2H, d, J=8.5 Hz, H3, H5), 6.10 (0. 38H, s, H30 , H50 ), 5.35 (1H, d, J=1.7 Hz, Rha-H1), 5.08 (1H, d, J=7.4 Hz, Glu-H1), 3.98 (1H, dq, J=6.2, 9.5 Hz, Rha-H5), 3.94 (1H, dd, J=1.7, 3.4 Hz, Rha-H2), 3.91 (1H, dd, J=2.3, 11.9 Hz, Glu-H6a), 3.73 (1H, dd, J=5.8, 11.9 Hz, Glu-H6b), 3.71 (1H, m, Glu-H3), 3.68 (1H, dd, J=7.4, 9.9 Hz, Glu-H2), 3.63 (1H, dd, J=3.4, 9.5 Hz, Rha-H3), 3.56 (1H, ddd, J=2.3, 5.8, 9.6 Hz, Glu-H5), 3.48 (1H, t, J=9.6 Hz, Glu-H4), 3.47 (1H, t, J=9.5 Hz, Rha-H4), 1.26 (3H, d, J=6.2 Hz, Rha-H6) ppm. 13C NMR d 207.1, 165.9 (C20 , C60 ), 165.2 (C40 ), 157.4 (C4), 134.3 (C1), 131.2 (C2, C6), 117.0 (C3, C5), 107.5 (C10 ), 102.6 (Rha-C1), 100.0 (Glu-C1), 97.3 (C30 , C50 ), 79.8 (Glu-C3), 78.7 (Glu-C5), 78.6 (Glu-C2), 74.9 (Rha-C4), 73.3 (Rha-C3), 72.9 (Rha-C2), 72.5 (Glu-C4), 70.2 (Rha-C5), 63.5 (Glu-C6), 19.4 (Rha-C6) ppm. FDMS m/z 610.2145 (M+Na+, C27H30D5O14Na requires 610.2160, isotopic composition d2:d3:d4:d5:d6:d7:d8:d9=16:54:89:100:83:51:21:8). LCMS-MS m/z 587
2 (M-H , isotopic distribution d3:d4:d5:d6:d7=5:31:86:100:14), 466 (ms ), 357, 277 (ms3). Conclusion Pd/C catalysed deuterium exchange using methanol-d1 as the deuterium source provides ready access to a variety of complex deuterated dihydrochalcones glycosides. As this labelling method is also amenable to the incorporation of tritium, it should be widely useful for studying biological activity of this in- teresting class of flavonoids. Copyright # 2006 John Wiley & Sons, Ltd. J Label Compd Radiopharm 2006; 49: 479–487  SYNTHESIS OF DEUTERATED DIHYDROCHALCONES 487 Acknowledgements We wish to thank Martin Hunt for FDMS and the Foundation for Research Science and Technology for financial support under Contract COX0220. References 1. Bohm BA. The minor flavanoids. In The Flavanoids Advances in Research since 1986, Harbourne JB (ed.). Chapman & Hall: London, 1993; 399–406. 2. Kamperdick C, Van NH, Van Sung TV. Phytochemistry 2002; 61(8): 991–994. 3. Kumar JK, Narender T, Rao MS, Rao PS, Toth G, Balazs B, Duddeck H. J Braz Chem Soc 1999; 109(4): 278–280. 4. Tanaka T, Uehara R, Nishida K, Kouno I. Phytochemistry 2004; 65: 1–7. 5. Ogawa K, Kawasaki A, Omura M, Yoshida T, Ikoma Y, Yano M. Phytochem- istry 2001; 57(5): 737–742. 6. Tomas-Barberan FA, Clifford MN. J Sci Food Agric 2000; 80(7): 1073–1080. 7. Hilt P, Schieber A, Yildirim C, Arnold G, Klaiber I, Conrad J, Beifuss U, Carle R. J Agric Food Chem 2003; 51(10): 2896–2899. 8. Yang W-M, Liu J-K, Qin X-D, Wu W-L, Chen Z-H. Z Naturforsch [C] 2004; 59c: 481–484. 9. Alvarado F, Crane RK. Biochim Biophys Acta 1964; 93: 116–135. 10. Rossetti L, Smith D, Shulman GI, Papachristou D, Defronzo RA. J Clin Investig 1987; 79: 1510–1515. 11. Starke A, Grundy S, McGarry JD, Unger RH. Proc Natl Acad Sci USA 1985; 82(5): 1544–1546. 12. Zhao H, Yakar S, Gavrilova O, Sun H, Zhang Y, Kim H, Setser J, Jou W, LeRoith D. Diabetes 2004; 53: 2901–2909. 13. Monge P, Solheim E, Scheline RR. Xenobiotica 1984; 14: 917–924. 14. Crespy V, Aprikian O, Morand C, Besson C, Manach C, Demigne C, Remesy C. J Nutr 2001; 131(12): 3227–3230. 15. Tuck KL, Tan H-W, Hayball PJ. J Label Compd Radiopharm 2000; 43: 817–823. 16. Yao J, Evilia RF. J Am Chem Soc 1994; 116(25): 11229–11233. 17. Hibbs MR, Yao J, Evilia RF. High Temp Mater Sci 1996; 36: 9–14. 18. Wa¨ha¨la¨ K, Rasku S. Tetrahedron Lett 1997; 38: 7287–7290. 19. Krishnamurty HG, Sathyanarayan S. Synthetic Commun 1989; 19(1 and 2): 119–123. 20. Rajagopal S, Spatola AF. J Org Chem 1995; 60(5): 1347–1355. 21. Matich AJ, Bunn BJ, Hunt MB, Rowan DD. Phytochemistry: http://www. sciencedirect.com/science?_ob=Mlmg&_imagekey=B6TH7-4J6W780-2-9&_cdi= 5275&_user=706881&_orig=search&_coverDate=02%2F07%2F2006&_sk= 999999999&_view=c&wchp=dGLbVIb-zSkWb&md5=1a1628cb1324f98fdb7a940 c419faf42&ie=/sdarticle.pdf 22. Markham KR, Geiger H. 1H nuclear magnetic resonance spectroscopy of flavonoids and their glycosides in hexadeuterodimethylsulfoxide. In The Flavonoids Advances in Research Since 1986, Harbourne JB (ed.). Chapman & Hall: London, 1993; 441–497. Copyright # 2006 John Wiley & Sons, Ltd. J Label Compd Radiopharm 2006; 49: 479–487