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Biotransformation of flavokawains A, B and C, chalcones from Kava (Piper methysticum), by human liver microsomes Katharina Zenger, Sara Agnolet, Bernd Schneider, and Birgit Kraus J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.5b01858 • Publication Date (Web): 30 Jun 2015 Downloaded from http://pubs.acs.org on July 8, 2015

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Page 1 of 40

Journal of Agricultural and Food Chemistry

Biotransformation of flavokawains A, B and C, chalcones from Kava (Piper methysticum), by human liver microsomes K. Zenger1, S. Agnolet2, B. Schneider2, B. Kraus1* 1

Pharmaceutical Biology, Institute of Pharmacy, University of Regensburg, Universitätsstraße 31, 93053 Regensburg, Germany

2

Max-Planck-Institute for Chemical Ecology, Beutenberg Campus, Hans-Knöll-Str. 8, 07745 Jena, Germany

* Corresponding author, (Tel: +49 941 9434494; Fax: +49 941 9434990; E-mail: [email protected]);

1

ACS Paragon Plus Environment


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1

Abstract

2

The in vitro metabolism of flavokawain A, B and C (FKA, FKB, FKC), methoxylated

3

chalcones from Piper methysticum, was examined using human liver microsomes.

4

Phase I and phase II (glucuronidation) metabolism as well as combined phase I+II

5

metabolism were studied. For identification and structure elucidation of microsomal

6

metabolites, LC–HRESIMS and NMR techniques were applied. Major phase I

7

metabolites were generated by demethylation in position C-4 or C-4' and

8

hydroxylation predominantly in position C-4, yielding FKC as phase I metabolite of

9

FKA and FKB, helichrysetin as metabolite of FKA and FKC, and cardamonin as

10

metabolite of FKC.

11

To an even greater extent, flavokawains were metabolized in presence of uridine

12

diphosphate (UDP) glucuronic acid by microsomal UDP-glucuronosyl transferases.

13

For

14

glucuronide, FKC-2'-O-glucuronide, FKC-4-O-glucuronide) were found as major

15

phase II metabolites. The dominance of generated glucuronides suggests a role of

16

conjugated chalcones as potential active compounds in vivo.

all

flavokawains,

monoglucuronides

(FKA-2'-O-glucuronide,

FKB-2'-O-

17 18 19

Keywords:

20

Piper methysticum, kava, flavokawain, cardamonin, metabolism, human liver

21

microsomes, glucuronides

22 23 24

2


Page 3 of 40


25

Introduction

26

Chalcones (1,3-diaryl-2-propen-1-ones) are open chain flavonoids that can be found

27

in a variety of pharmaceutically relevant plants like hops (Humulus lupulus) 1, licorice

28

(Glycyrrhiza glabra) 2, or willow (Salix sp.) 3, as well as in fruits and vegetables like

29

apples (Malus sp.) 4, citruses (Citrus sp.) 5 and tomatos (Lycopersicon esculentum) 6.

30

A broad range of biological activities has been described for chalcones, including

31

antioxidant 7,8, anti-inflammatory 9–11, chemopreventive 1,12, anti-cancer 13,14, and anti-

32

infective properties

33

possess a high potential for therapeutic application and may serve as lead

34

compounds for drug development 17.

35

Flavokawain A, B and C (FKA, FKB, FKC) are naturally occurring methoxylated

36

chalcones (Figure 1), that have been isolated from Piper methysticum FORST.

37

(Piperaceae), which is also known as Kava

38

Western Polynesia, but is nowadays cultivated nearly all over the Pacific Islands,

39

from Hawaii to Papua New Guinea. In these countries, the consumption of

40

beverages, based on water extracts of kava rhizome, has a long-standing tradition.

41

Kava is used as a relaxant at traditional social gatherings and in religious ceremonies

42

to achieve a higher level of consciousness. In addition, the beverage reduces fatigue,

43

relieves from anxiety and generates a state of well-being and a cheerful and sociable

44

attitude 20,21.

45

The consumption of kava has spread to the Western world where preparations of

46

kava were approved for the treatment of anxiety and nervous disorders such as

47

stress and restlessness

48

replaced by commercially available preparations made from acetonic or ethanolic

49

extracts to specifically extract kava lactones, that are considered to be the active

50

constituents

21,22

15,16

. Having significant pharmacological activities, chalcones

21

18,19

. The kava plant has its origin in

. However, the traditional kava rhizome water extract was

. In 2002, all kava containing preparations were banned from the 3



Page 4 of 40

51

German market as the safety of kava products had been questioned due to reported

52

hepatotoxic side effects

53

benefit-to-risk profile was assessed positively and the hepatotoxic potential of kava

54

was judged not to be verified sufficiently 25.

55

Kava lactones are the major active compounds found in kava. They are held

56

responsible for the anxiolytic and sedative effect of kava

57

pharmacological effects were described for the chalcones FKA, FKB and FKC, which

58

are contained in minor amounts in kava. All three flavokawains have been shown to

59

exhibit anti-cancer activity by inducing apoptosis in bladder cancer cells

60

suits

61

predominantly by men, correlates with low and uncustomary gender ratios of cancer

62

incidences (more cancer in women than men) in three kava drinking countries (Fiji,

63

Vanuatu, Western Samoa). Furthermore, FKA and FKB show anti-inflammatory

64

activity by suppressing the expression of inducible nitric oxide synthase and

65

cyclooxygenase-2 in lipopolysaccharide induced RAW 264.7 cells via the inhibition of

66

NF-κB pathway

67

FKB in mice 30,31.

68

Despite increasing data regarding bioactivity, to date there is no information about

69

metabolism of flavokawains. In the present study, we therefore analyzed the in vitro

70

metabolism of flavokawains using human liver microsomes. Different microsomal

71

incubation systems were applied to study phase I metabolism and phase II

72

glucuronidation as well as combined phase I and II metabolism. Metabolites were

73

identified by liquid chromatography–high resolution electrospray ionization mass

74

spectrometry (LC–HRESIMS) and nuclear magnetic resonance spectroscopy (NMR).

75

A metabolic profile of flavokawains is proposed.

reports

that

28,29

23,24

. However, this ban was cancelled in 2014, as the

consumption

of

traditional

21

. In addition, a variety of

kava beverage

26,27

. This

preparations,

. Moreover, Mohamad et al. proposed a nociceptive activity of

76 4


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77


Material and Methods

78 79

Chemicals and reagents

80

Glucose-6-phosphate sodium salt (G6P), glucose-6-phosphate dehydrogenase

81

(G6PDH), nicotinamide adenine dinucleotide phosphate sodium salt hydrate (NADP),

82

uridine 5‘-diphosphoglucuronic acid trisodium salt (UDPGA) and alamethicin (Ala)

83

from Trichoderma viride were obtained from Sigma-Aldrich (Taufkirchen, Germany).

84

Acetonitril (MeCN, LiChroSolv) and methanol (LiChroSolv) were obtained from Merck

85

(Darmstadt, Germany). All other used solvents were of pro analysis quality and

86

derived from Merck or Arcos.

87 88

Human liver microsomes

89

Frozen pooled liver microsomes (HMMC-PL, 0.5 mL) were purchased from Life

90

Technologies (Darmstadt, Germany). They derived from 50 adult donors of mixed

91

gender having a total protein concentration of 20 mg/mL and a cytochrome P450

92

content of 0.286 nmol/mg. Microsomes were stored at -80 °C until use. A detailed

93

certificate of analysis of used microsomes can be found on the provider’s homepage.

94 95

Test compounds

96

FKA, FKB, FKC and cardamonin were synthesized during previous work at the

97

Institute of Pharmacy, University of Regensburg, and showed no impurities in the

98

HPLC runs 7. For preparation of stock solutions, test compounds were first dissolved

99

in DMSO at a concentration of 10 mM and then diluted with EtOH to a concentration

100

of 1 mM.

101

5



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102

Microsomal incubation systems for phase I metabolism and phase II

103

glucuronidation

104

For phase I metabolism, a NADPH regeneration system was used. A typical 1 mL

105

incubation mix consisted of 0.5 mg liver microsomal protein, 3.3 mM MgCl2, 3.3 mM

106

G6P, 0.4 U/mL G6PDH, 1.3 mM NADP and 10 µM flavokawain or cardamonin in 0.1

107

M potassium phosphate buffer.

108

For phase II glucuronidation, the 1 mL incubation mix was composed of 0.5 mg

109

microsomal protein, 3.3 mM MgCl2, 2 mM UDPGA, 25 µg/mL Ala and 10 µM

110

flavokawain or cardamonin in 0.1 M potassium phosphate buffer.

111

Combined phase I and II metabolism was investigated using a 1 mL incubation

112

system consisting of 0.5 mg microsomal protein, 3.3 mM MgCl2, 3.3 mM G6P, 0.4

113

U/mL G6PDH, 1.3 mM NADP, 2 mM UDPGA, 25 µg/mL Ala and 10 µM flavokawain

114

or cardamonin in 0.1 M potassium phosphate buffer.

115

The reaction was started by addition of NADP or/and UDPGA. Incubation was carried

116

out in a shaken water bath at 37 °C for 60 min. Negative control incubations were

117

carried out without microsomes or without cofactors (NADP or UDPGA). Reactions

118

were terminated by addition of 1 mL ice-cold EtOH. Samples were vortexed for 5 min

119

and then centrifuged for 5 min at 14.000 rpm and 4 °C. Supernatants were subjected

120

to further analysis.

121

For NMR, sample size was increased to 5 mL and the concentration of chalcones

122

was raised to 100 µM due to lower sensitivity of NMR compared to MS. An incubation

123

time of 4 h was chosen to maximize transformation rate. Metabolic reactions were

124

stopped by addition of ice-cold EtOH. To improve storage stability, EtOH was

125

evaporated and samples were refilled with ultra-pure water. Then samples were

126

freeze-dried and stored at -20 °C until further analysis.

127 6


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128

Liquid

chromatography–high

resolution

electrospray

ionization

mass

129

spectrometry (LC–HRESIMS)

130

Samples for LC-HRESIMS analysis were prepared after 0 min and 60 min incubation.

131

Matrix samples without test compound referred as blank controls. Analysis was

132

performed with UHPLC Agilent 1290 infinity, DAD G4212A and MS Agilent 6540

133

UHD Q-TOF with negative electrospray ionization using dual ion source. Separation

134

was performed on a Thermo Accucore aQ column (C18, 50 x 2.1 mm, 2.6 µm) using

135

a gradient of 0.1% formic acid (solvent A) and 100% MeCN supplemented with 0.1%

136

formic acid (solvent B). Gradient: 0-10 min 0→98% B; 10-11 min 98% B; 11-11.1 min

137

98→0% B; 11.1-13 min 0% B; flow rate: 0.6 mL/min, injection volume: 5 µL, oven

138

temperature: 40 °C. Data analysis was performed with MassHunter software

139

(B.05.00, Agilent) using automatic mass spectrum integration.

140 141

HRESIMS of 5,7-dimethoxy-flavanone-4'-O-glucuronide

142

HRESIMS

143

recorded on a LC–MS/MS system consisting of an Ultimate 3000 series RSLC

144

(Dionex, Sunnyvale, CA, USA) system and an Orbitrap mass spectrometer (Thermo

145

Fisher Scientific, Bremen, Germany). HRESIMS data were analyzed using

146

XCALIBUR (Thermo Fisher Scientific, Waltham, MA, USA) software.

of

FKC

metabolite

5,7-dimethoxy-flavanone-4´-O-glucuronide

was

147 148

Isolation and structure elucidation of metabolites

149

Samples were dissolved with a minimum amount of HPLC solvent and HPLC

150

fractionation was performed using an Agilent HP1100 Series HPLC system (Agilent

151

Technologies, Waldbronn, Germany) equipped with binary pump G1312A, degasser

152

G1322A, autoinjector 1367A, column oven G1316, and G11315B diode array

153

detector controlled with ChemStationRev.A.08.04 (1008) software. Separation was 7



Page 8 of 40

154

performed on a LiChrosphere RP-18 column (5 µm, 250 x 4 mm, Merck KGaA,

155

Darmstadt, Germany) with a guard column (5 µm, 4 x 4 mm) using a linear binary

156

gradient of ultra-pure water containing 0.1% (v/v) trifluoroacetic acid (solvent A) and

157

MeCN (solvent B) with a flow rate of 0.8 mL/min. The gradient profile was 0-30 min

158

20%→80% B; 30-35 min 80% B; 35-37 min 80%→20% B; and column temperature

159

was set at 30 °C.

160

NMR spectra were recorded in MeOH-d4 with a Bruker Avance 500 NMR

161

spectrometer (Bruker-Biospin, Karlsruhe, Germany) operating at 500.13 MHz for 1H

162

and 125.75 MHz for 13C. The spectrometer was equipped with a 5 mm TCI cryoprobe

163

and capillary tubes (2 mm o.d.; 80 µL filling volume) were used for measuring NMR

164

spectra with standard Bruker pulse sequences. Tetramethylsilane was used as

165

internal chemical shift reference standard. The spectrometer was controlled by

166

TopSpin 3.1 software, which was also used for processing of the NMR spectra.

167

According to the limited amounts, only 1H NMR spectra were recorded of FKA

168

metabolites. In addition to 1H NMR spectra of FKB and FKC metabolites, 2D NMR

169

spectra (1H,1H-COSY; 1H,13C-HMBC; 1H,13C-HSQC) were recorded.

170 171

Results

172

To gain insight into the metabolism of flavokawains, FKA, FKB and FKC as well as

173

cardamonin were subjected to different microsomal incubation systems providing

174

phase I reactions or phase II glucuronidation. Furthermore, combined phase I and II

175

metabolism was studied by activation of phase I enzymes and UDP-glucuronosyl

176

transferases in one system. HPLC was used to confirm stability of the test

177

compounds in phosphate incubation buffer. In the control incubation systems without

178

cofactors or microsomes, no nonspecific metabolism or degradation was observed

179

(data not shown). 8


Page 9 of 40


180 181

Metabolites of FKA

182

Two phase I metabolites were found for FKA. According to retention times (tR) and

183

LC-MS data (Table 1), they were identified as helichrysetin (Heli) (tR = 4.02 min; m/z

184

[M-H]- 285.0766) and FKC (tR = 5.07 min; m/z [M-H]- 299.0920), with FKC being the

185

major phase I metabolite.

186

Phase II metabolism resulted in glucuronidation of FKA. Despite only one available

187

OH-group at C-2', two FKA glucuronides (tR = 3.38, m/z [M-H]- 489.1402 and tR =

188

3.50 min, m/z [M-H]- 489.1405) were detected, which are suggested to be E/Z-

189

isomers. Combined phase I and II metabolism led to the formation of a FKA

190

glucuronide (tR = 3.50 min, m/z [M-H]- 489.1402) as well as of two FKC glucuronides

191

(tR = 2.81, m/z [M-H]- 475.1238 and tR = 3.87 min, m/z [M-H]- 475.1239). Additionally,

192

hydroxylation and glucuronidation resulted in the generation of two OH-FKA

193

glucuronides (tR = 3.71, m/z [M-H]- 505.1345 and tR = 3.81 min, m/z [M-H]- 505.1345)

194

that were identified as minor metabolites. The exact positions of hydroxylation and

195

glucuronidation of major metabolites were established by NMR but could not be

196

determined for minor ones.

197 198

The aglycone part of the 1H NMR spectrum of the FKA glucuronides revealed two

199

sets of signals, which by means of the couplings constants and chemical shift

200

differences of H-α and H-β (Table 2) were clearly assignable to the suggested (E)-

201

and (Z)-isomers of the aglycone with JHα-Hβ = 15.9 Hz indicating the (E)-form being

202

the major one. The (Z)-form (JHα-Hβ = 12.5 Hz) might constitute an artifact that is

203

formed during incubation, sample processing, or isolation procedure because it was

204

detected again after separation of the peak at tR = 3.50 min by HPLC. Comparison of

205

the 1H NMR spectrum of the FKA glucuronides with the parent compound clearly 9



Page 10 of 40

206

shows a strong difference in the chemical shifts for H-3' and H-5' in ring B and for the

207

two olefinic protons α/β while the chemical shifts of the protons in ring A changed

208

only slightly (Table 2). This constitutes evidence of the glucuronide moiety being

209

attached to 2'-OH. According to the limited availability of the FKA metabolites and the

210

resulting poor signal-to-noise ratio,

211

assigned from the HSQC and HMBC spectra, but the obtained data of FKA-2´-O-

212

glucuronide ((E)-form: δ 95.3 (C-3’), 93.8 (C-5’), 131.8 (C-2/6), 115.6 (C-3/5), 55.7

213

(4’-OCH3), 56.2 (6’-OCH3), 56.0 (4-OCH3), 164.2 (C-4 and C-4’), and 160.8 (C-6’))

214

supported the suggested structure. The resonances of the glucuronide moieties

215

could not be completely assigned from the 1H NMR and 1H,1H COSY spectrum of the

216

mixtures of the (E)- and (Z)-isoform because of strong overlap of signals in the

217

spectral region of carbohydrate signals. A metabolism scheme of FKA is proposed in

218

Figure 2. Although, due to this overlap, the coupling constant of the H-1" signal was

219

invisible, the β-configuration was inferred from its chemical shift (δ 4.99) for both the

220

(E)- and (Z)-isomers, while a corresponding α-H-1" signal is expected at lower field

221

32

13

C chemical shifts could not be completely

.

222 223

Metabolites of FKB

224

For FKB, also FKC (tR = 5.07 min, m/z [M-H]- 299.0927) was detected as major

225

phase I metabolite (Table 3). In addition, cardamonin (tR = 5.17 min, m/z [M-H]-

226

269.0819) and 2',4',6'-trihydroxychalcone (tR = 4.48 min, m/z [M-H]- 255.0659) were

227

generated. The 2'-O-glucuronide of FKB (tR = 3.46 min, m/z [M-H]- 459.1297) was

228

found after phase II metabolism. Combined phase I and II metabolism also lead to

229

the formation of 2'-O-glucuronide of FKB and additionally to four further glucuronides.

230

Two of them (tR = 2.81 min, m/z [M-H]- 475.1239 and tR = 3.87 min, m/z [M-H]-

231

475.1241) were identified as 2'- and 4-O-glucuronide of FKC. As only two OH-groups 10


Page 11 of 40


232

are available on FKC, the other two generated glucuronides (tR = 3.15 min, m/z [M-

233

H]- 475.1239 and tR = 3.75 min, m/z [M-H]- 475.1241) probably are Z-isoforms of

234

those

235

glucuronidated (tR = 3.95 min, m/z [M-H]- 445.1123) either at position C-2' or C-4'.

metabolites.

Furthermore,

the

phase

I

metabolite

cardamonin

was

236 237

(E)-FKB-2'-O-β-glucuronide constitutes the major metabolite of FKB and its structure

238

was unambiguously identified by 1H NMR, COSY, HSQC, and HMBC. Comparison of

239

the 1H NMR spectrum of the major FKB metabolite with that of the parent compound

240

clearly shows the slightly changed chemical shifts for the protons in ring B while a

241

strong change in the chemical shift position occurs for H-3' and H-5' and for the two

242

olefinic protons α/β (Table 4), indicating structural modification near ring A. The large

243

coupling constant JHα-Hβ = 15.9 Hz indicated that the E-configuration at the α/β

244

double bond has been retained in the metabolite. The signal of the proton (δ 5.02) at

245

the anomeric C-1'' displays the coupling constant of JH1"-H2" = 7.8 Hz, typical of β-

246

configuration. This signal also shows a reduced signal-to-noise ratio due to its

247

proximity to the suppressed residual water signal. The 1H NMR signal of H-5'' (δ 3.85)

248

is overlapping with the 4'-methoxy signal (δ 3.85). The remaining signals of the β-

249

glucuronide moiety were assigned by the COSY spectrum even if some signals

250

overlapped. 13C resonances were obtained from HSQC and HMBC spectra (Table 4).

251

13

252

detected (n.d.) because of low signal-to-noise ratio of the HMBC spectrum. Again

253

signals of (Z)-FKB-2'-O-β-glucuronide (data not shown) were detected as a minor

254

component accompanying the major (E)-from. A metabolism scheme of FKB is

255

proposed in Figure 3.

C NMR chemical shifts of the quaternary C-2' and the COOH carbon atom were not

256 257

Metabolites of FKC 11



Page 12 of 40

258

LC-MS data of FKC metabolism samples revealed Heli (tR = 4.03 min, m/z [M-H]-

259

285.0767) as major phase I metabolite (Table 5). In addition, hydroxylation at

260

undetermined positions generated two OH-FKC (tR = 4.00 min, m/z [M-H]- 315.0872

261

and tR = 4.52 min, m/z [M-H]- 315.871). Two major monoglucuronides (tR = 2.81 min,

262

m/z [M-H]- 475.1245 and tR = 3.86 min, m/z [M-H]- 475.1248) and one minor

263

glucuronide of FKC (tR = 2.71 min, m/z [M-H]- 475.1244) were detected after phase II

264

metabolism. Combined phase I and II metabolism resulted in the generation of one

265

Heli-glucuronide (tR = 3.03 min, m/z [M-H]- 461.1085) and three different

266

glucuronides of OH-FKC (tR = 2.83 min, m/z [M-H]- 491.1183; tR = 3.18 min, m/z [M-

267

H]- 491.1188; tR = 3.72 min, m/z [M-H]- 491.1187).

268 269

The two major monoglucuronides of FKC (tR = 2.81 and 3.86 min) were identified by

270

NMR spectroscopy as (E)-FKC-4-O-β-glucuronide and (E)-FKC-2'-O-β-glucuronide,

271

the minor glucuronide (tR = 2.71 min) as 5,7-dimethoxy-flavanone-4'-O-β-glucuronide

272

(Figure 4). Comparing the 1H NMR spectrum of FKC-4-O-β-glucuronide with that of

273

the parent compound shows the slightly changed chemical shifts for H-3' and H-5' in

274

ring A and the olefinic protons α/β. The chemical shifts of H-2/6 and H-3/5 changed

275

strongly from δ 7.51 and δ 6.82 in the spectrum of FKC to δ 7.62 and δ 7.15 in the

276

spectrum of the glucuronide, indicating a substitution in ring B (Table 6). The large

277

coupling constant of JHα-Hβ = 15.5 Hz indicated E-configuration of the α/β double

278

bond. The signal of H-1" (δ 5.05) shows a reduced signal-to-noise ratio due to its

279

proximity to the suppressed residual water signal and a spin-spin coupling JH1"-H2" =

280

7.7 Hz characteristic of β-configuration at the anomeric center. The remaining signals

281

of the β-glucuronide moiety were putatively assigned based on 1H NMR data

282

matching with corresponding glucuronide signals of the other metabolites.

12


Page 13 of 40


283

Different from FKC-4-O-β-glucuronide, comparison of the 1H NMR spectrum of FKC-

284

2'-O-β-glucuronide with that of the parent compound reveals only little chemical shift

285

changes for H-2/6 and H-3/5 in ring B while a strong change in the chemical shifts

286

occurs for H-3' and H-5' and for the two olefinic protons α/β (Table 6). This finding

287

resembles that of major FKA- and FKB-2'-O-β-glucuronides described above. Thus,

288

the substitution must have occurred in the 2'-position. Well resolved signals of the

289

glucuronide moiety with very good signal-to-noise ratio were detected in the 1H NMR

290

spectrum of FKC-2'-O-β-glucuronide, allowing the precise determination of the JH-H

291

coupling constants. This included JH1"-H2" = 7.7 Hz, indicating β-configuration at the

292

anomeric center of the sugar unit. In addition, the 2D NMR experiments (1H,1H-

293

COSY, HSQC, HMBC) enabled assignment of all 1H and

294

HMBC correlation between H-5'' (δ 3.97) and the

295

chemical shift for carboxylic carbons, proves the carbohydrate unit to be a

296

glucuronide. The HMBC cross signal between H-1" (δ 7.15) and C-2' (δ 157.8) clearly

297

proved the attachment of the carbohydrate to the 2'-O-position. HMBC also enabled

298

assignment of 1H resonances, such as those of the methoxy groups or protons H-3'

299

and H-5', that would be otherwise potentially interchangeable.

13

13

C chemical shifts. An

C signal at δ 171.9, a typical

300 301

For 5,7-dimethoxy-flavanone-4'-O-β-glucuronide, the disappearance of the signals of

302

the olefinic α/β protons in the 1H NMR spectrum together with the appearance of new

303

resonances of H-2 (δ 5.43) and H-3 (δ 3.04 / δ 2.72) enables the assignment of the

304

aglycone skeleton as a flavanone (Figure 4). The molecule was fully identified by

305

means of 1D and 2D NMR spectroscopic data. In addition to the aglycone signals

306

(Table 7), the 1H NMR spectrum displays signals of the glucuronide unit with some

307

overlap of the signals of H-2'', H-3'' and H-4'' and a doublet of the β-configured H-1"

13



Page 14 of 40

308

(δ 4.98; JH1"-H2" = 7.5 Hz). HMBC correlation between H-1" and C-4' (δ 158.9)

309

assigned the β-glucuronide unit to the hydroxyl group in p-position of ring B.

310

Interestingly, the signals of H-3β (δ 2.72) appear as a two double doublets (dd) with

311

integral values corresponding to 0.5 protons each. This could be explained by the

312

assumption, that the compound is a racemate, which consists of two diastereomers

313

with opposite configuration at C-2. The signals of H-2 of the 2R- and the 2S-form as

314

well as the signals of the H-3α at δ 3.04 are isochronic. Only the dd signals of H-3β

315

appear at slightly different chemical shifts (∆δ ~0.01 ppm). If this assumption is

316

correct, the 5,7-dimethoxy-flavanone-4'-O-β-glucuronide is probably generated by a

317

non-enzymatic chemical reaction and therefore an artifact. The HRESIMS spectrum

318

(positive ionization) confirmed the structure. In Figure 4 a metabolic pathway for FKC

319

is proposed.

320 321

Metabolites of cardamonin

322

For additional information, also the chalcone cardamonin, which was found as an

323

phase I metabolite of FKB, was subjected to phase I and II metabolism as well as

324

combined phase I + II metabolism.

325

LC-MS data of cardamonin metabolism samples are depicted in Table 8. Three

326

hydroxylated metabolites of cardamonin were found after phase I metabolism, one of

327

them was identified as Heli (tR = 4.03 min, m/z [M-H]- 285.0763). For the other two

328

metabolites (tR = 4.43 min, m/z [M-H]- 285.0763 and tR = 4.52 min, m/z [M-H]-

329

285.0762), the exact positions for the OH-group could not be determined.

330

After phase II metabolism, three glucuronides of cardamonin (tR = 2.67 min, m/z [M-

331

H]- 445.1141; tR = 3.01 min, m/z [M-H]- 445.1138; tR = 3.95 min, m/z [M-H]- 445.1140)

332

were detected. As only two free OH-groups are available, a third corresponding mass

333

suggests the presence or formation of isomers. Additionally to the cardamonin 14


Page 15 of 40


334

glucuronides, one glucuronide of OH-cardamonin (tR = 3.58 min, m/z [M-H]-

335

461.1081) was found after combined metabolism. Figure 5 suggests a metabolic

336

pathway of cardamonin.

337 338

Discussion

339

In this study, we characterized the in vitro metabolism of FKA, FKB, FKC and

340

cardamonin using human liver microsomes. In phase I metabolism, demethylation in

341

position C-4 or C-4' and hydroxylation predominantly in position C-4 occurred.

342

Demethylation at C-4 on the B-ring of FKA as well as hydroxylation at C-4 of FKB

343

lead to the formation of FKC, being the major phase I metabolite. Further

344

demethylation of FKC at C-4' on the A-ring generated Heli. Demethylation of FKB at

345

C-4' led to the generation of cardamonin, hydroxylation of cardamonin at C-4 to the

346

formation of Heli. Moreover, other monohydroxylated minor metabolites of FKA and

347

cardamonin were found, but the position of hydroxylation could not be determined by

348

mass spectrometric methods.

349

He et al. investigated the CYP450 dependent metabolism of cardamonin

350

previously 33.

351

fragmentation. The exact position of hydroxylation on the B-ring was not determined.

352

However, the authors suggested C-4 being the most likely position. Kohno et al.

353

already described this position as preferential for hydroxylation of chalcones

354

According to this, in the study of He et al. 4-OH-cardamonin would correspond to

355

Heli, which has also been found in our study. Furthermore, they suggested

356

hydroxylation in β-position of the unsaturated ketone, which might correspond to

357

another hydroxylated derivative (tR = 4.43 or 4.52 min) of our study. In contrast to our

358

findings, no third hydroxylated metabolite of cardamonin was described 33.

They

identified

two

monohydroxylated

metabolites

by

MS

34

.

15



Page 16 of 40

359

The compounds were even more extensively metabolized by microsomal UDP-

360

glucuronosyl transferases in the presence of UDPGA in phase II samples.

361

Corresponding monoglucuronides were detected for all test compounds as the major

362

metabolites of phase II and combined metabolism. The structures of the major

363

flavokawain glucuronides were elucidated by NMR.

364

After glucuronidation, two metabolite masses corresponding to FKA-glucuronides

365

were detected, despite only one available OH-group at C-2' of FKA. This can be

366

explained by the occurrence of the major E- and the minor Z-isomer. For both, 1H

367

NMR signals were detected. The occurrence of three glucuronides of cardamonin,

368

despite only two free OH groups, also suggests the formation of cardamonin E/Z-

369

isomers.

370

In addition to the 2'-O- and 4-O-chalcone-mono-β-glucuronides of FKC, also the

371

corresponding 5,7-dimethoxyflavanone-4'-O-β-glucuronide was identified by LC–

372

NMR. The flavanone-glucuronide is most likely formed by a non-enzymatic chemical

373

reaction and might be an artifact similar to the (Z)-isoforms of FKA, FKB and

374

cardamonin metabolites.

375

As FKC is a major phase I metabolite of FKA and FKB, and Heli is a metabolite of

376

FKC as well as of the FKB-metabolite cardamonin, the metabolism of all three

377

flavokawains (and cardamonin) is closely linked to each other. Therefore, we propose

378

a combined metabolism pathway for the flavokawains in Figure 6.

379 380

Although the metabolic conversions occurring in our in vitro system are very likely to

381

also occur in vivo, it is likewise almost certain, that they are only constituting a part of

382

the entire metabolization that is happening in the body. Therefore, the extent of

383

formation of conjugated metabolites may even be underestimated by the use of

384

microsomes, as phase II reactions in this system are limited to glucuronidation 16


Page 17 of 40


385

reactions only. As a result, the absolute amount of conjugated metabolites in vivo

386

may even be higher than what we observed in vitro. To include all possible phase II

387

metabolites, liver cell models and in vivo metabolism systems could be used for an

388

additional metabolism analysis. However, the fact that glucuronides dominate over

389

phase I metabolites underlines the important role of conjugated chalcone metabolites

390

as presumable in vivo active principle. This was likewise shown for various flavonoids

391

and chalcones, as e.g. xanthohumol 35.

392

Nevertheless, as far as the biological activity or toxicity of chalcones is concerned, to

393

date conjugated metabolites are not routinely included in testing.

394

Looking ahead, assessment of toxicity and pharmacological characterization of

395

flavokawain glucuronides and other phase II metabolites is mandatory and will be

396

conducted to evaluate their contribution and relevance for the bioactivity of the parent

397

compounds.

398 399

Abbreviations used

400

Ala, alamethicin; EtOAc, ethyl acetate; FKA, flavokawain A; FKB, flavokawain B;

401

FKC, flavokawain C; dd, double doublets; G6P, glucose-6-phosphate; G6PDH,

402

glucose-6-phosphate dehydrogenase; Gluc, glucuronic acid; Heli, helichrysetin;

403

NMR,

404

chromatography–high resolution electrospray ionization mass spectrometry; MeCN,

405

acetonitrile; NADP, nicotinamide adenine dinucleotide phosphate; NF-κB, nuclear

406

factor kappa-B; tR, retention time; UDP, uridine diphosphate; UDPGA, uridine 5'-

407

diphosphoglucuronic acid;

nuclear

magnetic

resonance

spectroscopy;

LC–HRESIMS,

liquid

408 409 410

Acknowledgements 17



Page 18 of 40

411

We thank Jörg Heilmann (Institute of Pharmacy, University of Regensburg) for fruitful

412

discussions and scientific support.

413 414

Supporting Information Available:

415

Corresponding MS and NMR spectra of the determined metabolites.

416

This material is available free of charge via the Internet at http://pubs.acs.org.

417

18


Page 19 of 40

418


References:

419 420

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pro-Inflammatory Mediators in Activated RAW 264.7 Cells and Whole Blood.

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Eur. J. Pharmacol. 2006, 538, 188–194.

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Chalcones potentialities as Antiproliferative and Antiresistance Agents.

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Hop (Humulus lupulus L.) in Comparison with Activities of Other Hop

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Constituents and Xanthohumol Metabolites. Mol Nutr Food Res 2005, 49, 827–

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Chromatography-Mass Spectrometry in Phytochemical Analysis of Kava (Piper

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Potentially Involved in Hepatotoxicity: A Review. Chem. Res. Toxicol. 2011, 24,

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Tumor Growth in Mice. Cancer Res. 2005, 65, 3479–3486.

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Chalcone Flavokawain A Differ in Bladder Cancer Cells with Wild-Type versus

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Mutant p53. Cancer Prev Res (Phila) 2008, 1, 439–451.

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H.; Chien, S.-C.; Wang, S.-Y. Anti-Inflammatory Activity of Flavokawain B from

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Alpinia pricei hayata. J. Agric. Food Chem. 2009, 57, 6060–6065.

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and COX-2 Expression by Flavokawain A via Blockade of NF-κB and AP-1

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Activation in RAW 264.7 Macrophages. Food Chem. Toxicol. 2013, 58, 479–

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Ong, H. M.; Zareen, S.; Akira, A.; Israf, D. A.; Lajis, N.; et al. Possible

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Participation of Nitric Oxide/cyclic Guanosine Monophosphate/protein Kinase

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C/ATP-Sensitive K(+) Channels Pathway in the Systemic Antinociception of

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Flavokawin B. Basic Clin. Pharmacol. Toxicol. 2011, 108, 400–405.

542 543

(31) Mohamad, A. S.; Akhtar, M. N.; Zakaria, Z. A.; Perimal, E. K.; Khalid, S.; Mohd,

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P. A.; Khalid, M. H.; Israf, D. A.; Lajis, N. H.; Sulaiman, M. R. Antinociceptive

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Activity of a Synthetic Chalcone, Flavokawin B on Chemical and Thermal

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Models of Nociception in Mice. Eur. J. Pharmacol. 2010, 647, 103–109.

547 548

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Nuclear Magnetic Resonance Spectra of All Positional Isomers of Methyl

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Mono-O-Tetradecanoyl-α- and β-D-Glucopyranosides. Chem. Pharm. Bull.

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1980, 28, 208–219.

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(33) He, Y.-Q.; Yang, L.; Liu, Y.; Zhang, J.-W.; Tang, J.; Su, J.; Li, Y.-Y.; Lu, Y.-L.;

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Wang, C.-H.; Yang, L.; et al. Characterization of Cardamonin Metabolism by

555

P450 in Different Species via HPLC-ESI-Ion Trap and UPLC-ESI-Quadrupole

556

Mass Spectrometry. Acta Pharmacol. Sin. 2009, 30, 1462–1470.

557 558

(34) Kohno, Y.; Kitamura, S.; Sanoh, S.; Sugihara, K.; Fujimoto, N.; Ohta, S.

559

Metabolism of the Alpha,beta-Unsaturated Ketones, Chalcone and Trans-4-

560

Phenyl-3-Buten-2-One, by Rat Liver Microsomes and Estrogenic Activity of the

561

Metabolites. Drug Metab. Dispos. 2005, 33, 1115–1123.

562 563

(35) Jirásko, R.; Holcapek, M.; Vrublová, E.; Ulrichová, J.; Simánek, V. Identification

564

of New Phase II Metabolites of Xanthohumol in Rat in Vivo Biotransformation

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of Hop Extracts Using High-Performance Liquid Chromatography Electrospray

566

Ionization Tandem Mass Spectrometry. J Chromatogr A 2010, 1217, 4100–

567

4108.

568 569 570

24


Page 25 of 40


571

Figure captions

572

Figure 1. Chemical structures of FKA, FKB and FKC.

573 574

Figure 2. Proposed metabolic pathway of FKA by human liver microsomes.

575

Structures of metabolites that could not be elucidated completely are depicted in

576

smaller size.

577 578

Figure 3. Proposed metabolic pathway of FKB by human liver microsomes.

579


580

smaller size.

581 582

Figure 4. Proposed metabolic pathway of FKC by human liver microsomes.

583


584

smaller size.

585 586

Figure 5. Proposed metabolic pathway of cardamonin by human liver microsomes.

587


588

smaller size.

589 590

Figure 6. Proposed combined metabolism scheme for FKA, FKB, FKC and

591

cardamonin. Structures of metabolites that could not be elucidated completely are

592

depicted in smaller size.

593 594

25



Page 26 of 40

Tables

Table 1. High Resolution LC-MS Data of FKA Samples Using Positive and Negative Electrospray Ionization.a

detected metabolites of FKA

tR

+

m/z [M+H]+ calculated

m/z [M-H]found

m/z [M-H]calculated

M formula

[min]

m/z [M+H] found

FKA

6.18

315.1234

315.1227

-

-

C18H18O5

Ph I

4.02 5.07

301.1073

301.1071

285.0766 299.0925

285.0768 299.0925

C16H14O5 C17H16O5

Ph II

3.38 3.50

491.1150 491.1151

491.1548 491.1548

489.1402 489.1405

489.1402 489.1402

C24H26O11 C24H26O11

Ph I+II

2.81 3.50 3.71 3.81 3.87 5.07

477.1389 491.1549 477.1391 301.1072

477.1391 491.1548 477.1391 301.1071

475.1238 489.1399 505.1345 505.1345 475.1239 299.0920

475.1246 489.1402 505.1351 505.1351 475.1246 299.0925

C23H24O11 C24H26O11 C24H26O12 C24H26O12 C23H24O11 C17H16O5

a

Major metabolites are written in bold. Corresponding mass spectra can be found in

SI.

26


Page 27 of 40


Table 2.

1

H-NMR Data (500 MHz, MeOH-d4) of FKA, (E)- and (Z)-FKA-2'-O-

glucuronide.

FKA

FKA-2'-O-glucuronide (E)-form (major)

1

1

(Z)-form (minor) 1

#

H δ (m, int., J [Hz ])

H δ (m, int., J [Hz])


3'

6.11 (d, 1H, J = 2.3)

6.57 (d, 1H, J = 2.3)

6.48 (d, 1H, J = 2.3)

5'

6.09 (d, 1H, J = 2.3)

6.36 (d, 1H, J = 2.3)

6.15 (d, 1H, J = 2.3)

α

7.71 (d, 1H, J = 15.9)

6.96 (d, 1H, J = 15.9)

6.39 (d, 1H, J = 12.5)

β 2/6

7.81 (d, 1H, J = 15.9)

7.39 (d, 1H, J = 15.9)

6.88 (d, 1H, J = 12.5)

7.60 (2H, d, J = 8.7)

7.58 (2H, m)

7.56 (2H, m)

3/5

6.98 (2H, d, J = 8.7)

6.95 (3H, m)

6.81 (3H, m)

4'-OCH3

3.95 (3H, s)

3.83 (3H, s)

3.79 (3H, s)

6'-OCH3

3.84 (3H, s)

3.78 (3H, s)

3.73 (3H, s)

4-OCH3

3.85 (3H, s)

2''

3.85 (3H, s) 4.99 (overlap with residual HDO signal) 3.38

3.80 (3H, s) n.d. (overlap with HDO signal) n.d.

3''

3.5 – 3.6 (m)

3.5 – 3.6 (m)

4''

n.d. n.d. (overlap with OCH3 signals)

n.d. n.d. (overlap with OCH3 signals)

1''

5''

27



Page 28 of 40

Table 3. High Resolution LC-MS Data of FKB Samples Using Positive and Negative Electrospray Ionization.a

detected metabolites of FKB

tR [min]

m/z [M+H]+ found


m/z [M-H]found

m/z [M-H]calculated

M formula

FKB

6.26

285.1125

285.1121

-

-

C17H16O4

Ph I

4.48 5.07 5.17

301.1070 -

301.1071 -

255.0659 299.0927 269.0819

255.0663 299.0925 269.0819

C15H12O4 C17H16O5 C16H14O4

Ph II

3.46

461.1445

461.1442

459.1297

459.1297

C23H24O10

Ph I+II

2.81 3.15 3.46 3.75 3.87 3.95 5.07

461.1441 477.1388 -

461.1442 477.1391 -

475.1239 475.1239 459.1293 475.1241 475.1241 445.1123 299.0920

475.1246 475.1246 459.1297 475.1246 475.1246 445.1140 299.0925

C23H24O11 C23H24O11 C23H24O10 C23H24O11 C23H24O11 C22H22O10 C17H16O5

a


SI.

28


Page 29 of 40


Table 4. 1H-NMR Data (500 MHz, MeOH-d4) of FKB and 1H- and 13C-NMR Data (500 MHz for 1H and 125 MHz for 13C, MeOH-d4) of (E)-FKB-2'-O-glucuronide.

FKB 1

#

(E)-FKB-2'-O-glucuronide 1

H

δ (m, int., J [Hz])

H


13

C

δ

1'

113.5

2'

n.d.

3'

6.12 (d, 1H, J = 2.3)

6.55 (d, 1H, J = 2.0)

164.1

4' 5'

95.3

6.10 (d, 1H, J = 2.3)

6.37 (d, 1H, J = 2.0)

93.8

6'

160.1

C=O

196.0

α

7.72 (d, 1H, J = 15.9)

7.09 (d, 1H, J = 15.9)

129.5

β 1

7.92 (d, 1H, J = 15.9)

7.42 (d, 1H, J = 15.9)

145.7

2-6

7.65 (2H, m)

3-5

7.42 (3H, m)

136.2

4

7.62 (2H, m) 7.39 (3H, m)

129.5 129.9 131.6

4'-OCH3

3.95 (3H, s)

3.85 (3H, s)

55.8

6'-OCH3

3.84 (3H, s)

3.79 (3H, s)

56.1

1''

5.02 (d, 1H, J = 7.8)

102.5

2''

3.38 (m, 1H)

74.6

3'' 4'' 5''

3.49 (m, 2H) 3.85 (overlap with OCH3)

77.3 73.3 76.3 n.d.

COOH

29



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Table 5. High Resolution LC-MS Data of FKC Samples Using Positive and Negative Electrospray Ionization.a

detected metabolites of FKC

tR [min]

m/z [M+H]+ found


m/z [M-H]found

m/z [M-H]calculated

M formula

FKC

5.07

301.1071

310.1070

299.0926

299.0925

C17H16O5

Ph I

4.00 4.03 4.52

287.0911 317.1019

287.0914 317.1019

315.0872 285.0767 315.0871

315.0874 285.0768 315.0874

C17H16O6 C16H14O5 C17H16O6

Ph II

2.71 2.81 3.86

477.1389 477.1392 477.1395

477.1391 477.1391 477.1391

475.1244 475.1245 475.1248

475.1246 475.1246 475.1246

C23H24O11 C23H24O11 C23H24O11

Ph I+II

2.71 2.81 2.83 3.03 3.18 3.72 3.86

477.1391 463.1234 477.1390

477.1391 463.1235 477.1390

475.1241 475.1241 491.1183 461.1085 491.1188 491.1187 475.1241

475.1241 475.1241 491.1195 461.1089 491.1195 491.1195 475.1241

C23H24O11 C23H24O11 C23H24O12 C22H22O11 C23H24O12 C23H24O12 C23H24O11

a

High resolution LC-MS data of FKC samples using positive and negative

electrospray ionization.

30


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Table 6. 1H-NMR Data (500 MHz, MeOH-d4) of FKC, FKC-4-O-glucuronide and FKC2'-O-glucuronide and 13C-NMR Data (125 MHz, MeOH-d4) of FKC-2'-O-glucuronide.

FKC

FKC metabolites (E)-FKC-4-Oglucuronide

1

#


1


(E)-FKC-2'-O-glucuronide 1


13

C δ

1'

113.7

2'

157.8

3'

6.10 (d, 1H, J = 2.3)

6.12 (d, 1H, J = 2.3)

6.49 (d, 1H, J = 2.0)

163.8

4' 5'

95.1

6.09 (d, 1H, J = 2.3)

6.10 (d, 1H, J = 2.3)

6.38 (d, 1H, J = 2.0)

93.8

6'

160.0

C=O

196.4

α β

7.71 (d, 1H, J = 15.6) 7.77 (d, 1H, J = 15.6)

7.71 (d, 1H, J = 15.5) 7.84 (d, 1H, J = 15.5)

6.89 (d, 1H, J = 16.0) 7.34 (d, 1H, J = 16.0)

126.4 147.4 127.2

1 2/6

7.51 (2H, d, J = 8.7)

7.62 (2H, d, J = 8.7)

7.48 (2H, d, J = 8.9)

131.6

3/5

6.82 (2H, d, J = 8.7)

7.15 (2H, d, J = 8.7)

6.80 (2H, d, J = 8.9)

116.6 161.5

4 4'-OCH3

3.94 (3H, s)

3.95 (3H, s)

3.85 (3H, s)

55.7

6'-OCH3

3.84 (3H, s)

3.85 (3H, s)

3.78 (3H, s)

56.1

1''

5.05 (d, 1H, J = 7.7)

102.4

2''

3.52 (2H, m)

5.05 (d, 1H, J = 7.7) 3.37 (dd, 1H, J = 7.7, 9.1) 3.46 (dd, 1H, J = 9.1, 9.1) 3.56 (dd, 1H, J =9.6, 9.1) 3.97 (d, 1H J = 9.6)

3'' 4''

3.65 (m)

5''

3.93 (m)

74.2 76.9 72.5 76.3 171.9

COOH

31



Table 7. 1H- and

13

Page 32 of 40

C-NMR Data of 5,7,-Dimethoxyflavanone-4'-O-glucuronide (500

MHz for 1H and 125 MHz for 13C, MeOH-d4).

5,7-dimethoxyflavanone-4'-Oglucuronide 1

13

H

#


δ

2

5.43 (dd, 1H, J = 12.8, 3.0)

79.9

3

3.04 (dd, 1H, J = 16.4, 12.8) 2.72 (dd, 1H, J = 16.4, 3.0)a

46.2

4

192.4

5

163.6

6

6.20 (d, 1H, J = 2.0)

8

93.7 168.3

7 6.22 (d, 1H, J = 2.0)

94.9

9

167.5

10

106.3

1'

134.2

2'/6'

7.44 (2H, d, J = 8.7)

128.7

3'/5'

7.14 (2H, d, J = 8.7)

117.8

4' 5- and 7OCH3 1'' 2'' 3''

158.9 3.84 (6H, s)

56.2

4.98 (d, 1H, J = 7.5)

102.3

3. 51 (m, 2H)

74.6 77.3

4''

3.59 (m, 1H)

73.1

5''

3.92 (d, 1H, J = 9.6)

76.3

COOH

a

C

173.5

Two dd at δ 2.723 and δ 2.717 with identical coupling constants indicate (2S)- and

(2R)-diastereomers.

32


Page 33 of 40


Table 8. High Resolution LC-MS Data of Cardamonin Samples Using Positive and Negative Electrospray Ionization.a

detected metabolites of cardamonin

tR [min]

m/z [M+H]+ found


m/z M-H]found

m/z [M-H]calculated

formula [M]

cardamonin

5.17

271.0970

271.0965

269.0820

269.0819

C16H14O4

Ph I

4.03 4.43 4.52

287.0915 287.0917 287.0917

287.0914 287.0914 287.0914

285.0763 285.0763 285.0762

285.0768 285.0768 285.0768

C16H14O5 C16H14O5 C16H14O5

Ph II

2.67 3.01 3.95

447.1286 447.1282 447.1287

447.1286 447.1286 447.1286

445.1141 445.1138 445.1140

445.1140 445.1140 445.1140

C22H22O10 C22H22O10 C22H22O10

Ph I+II

2.67 3.00 3.58 3.95

447.1284 447.1283 463.1232 447.1287

447.1284 447.1283 463.1235 447.1286

445.1135 445.1136 461.1081 445.1135

445.1140 445.1140 461.1089 445.1140

C22H22O10 C22H22O10 C22H22O11 C22H22O10

a


SI.

33



Page 34 of 40

Figures

Figure 1

34


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Figure 2

35



Page 36 of 40

Figure 3

36


Page 37 of 40


Figure 4

37



Page 38 of 40

Figure 5

38


Page 39 of 40


Figure 6

39



Page 40 of 40

Graphic for table of content

40


Biotransformation of Flavokawains A, B, and C ... - ACS Publications

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