Construction and Application of a Database for a Five-Dimensional

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Construction and Application of a Database for a Five Dimensional Identification of Natural Compounds in Garcinia species by means of UPLC-ESI-TWIMS-TOF-MS–Introducing Gas Phase Polyphenol Conformer Drift Time Distribution Intensity Ratios Timo D. Stark, Josef Ranner, Benedikt Stiglbauer, Patrick Weiss, Sofie Stark, Onesmo B. Balemba, and Thomas Hofmann J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b06157 • Publication Date (Web): 21 Dec 2018 Downloaded from http://pubs.acs.org on January 2, 2019

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Journal of Agricultural and Food Chemistry

1

Construction and Application of a Database for a Five

2

Dimensional Identification of Natural Compounds in

3

Garcinia species by means of UPLC-ESI-TWIMS-TOF-

4

MS–Introducing Gas Phase Polyphenol Conformer Drift

5

Time Distribution Intensity Ratios

6 7

Timo D. Stark,§* Josef Ranner,§ Benedikt Stiglbauer,§ Patrick Weiss,§

8

Sofie Stark,§ Onesmo B. Balemba,$ and Thomas Hofmann§

9 10

§Lehrstuhl

11

Universität München, Lise-Meitner-Str. 34, 85354 Freising, Germany

12

$Department

für Lebensmittelchemie und Molekulare Sensorik, Technische

of Biological Sciences, University of Idaho, Moscow, ID, USA

13 14 15 16 17 18 19

*

To whom correspondence should be addressed

20

PHONE

+49-8161-71-2911

21

FAX

+49-8161-71-2949

22

E-MAIL

[email protected]

23

1

ACS Paragon Plus Environment


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ABSTRACT

26

34 reference compounds from G. buchananii were analyzed by means of

27

UPLC-ESI-IMS-TOF-MS to build a database consisting of retention time,

28

accurate m/z of precursors and fragment ions as well as rotationally averaged

29

collision cross-sectional area (CCS).

30

compounds analyzed in bark extract in different concentrations and solvent

31

systems showed excellent intra- as well as interday precision (RSD ≤0.9%).

32

The established database was applied on different organs of G. buchananii as

33

well as G. kola, G. mangostana and G. cambogia enabling a fast and reliable

34

identification of these natural bioactives. For several compounds more than

35

one drift time species could be highlighted which we propose to be hydrogen

36

bond stabilized rotational isomers transferred from solution to gas phase. We

37

used all CCS values of one compound, and we propose to add also the

38

intensity ratio of the conformers as a new and additional characteristic

39

compound

40

applications to reduce dereplication and false positives and strengthen the

41

identification.

parameter

in

The CCS value of six selected

compound

identification/screening/database

42 43 44

KEYWORDS:

Ion

mobility,

CCS,

CCS

intensity

45

polyphenols, GB-2 7''-O-β-D-glucopyranosyl-6''-malonic acid, UNIFI database,

46

drift time

47

2


ratios,

conformers,

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INTRODUCTION

50

Ion mobility spectrometry (IMS) is a rapid and sensitive technique to separate

51

complex mixtures of (bio)molecules prior to mass spectrometry (MS). Strong

52

benefits arise between these two complementary methods as the information

53

about gas phase ions were significantly increased. IM is able to separate ions

54

from small molecules (< 500 Da) up to megadalton protein complexes based

55

on their differential mobility through a buffer gas, thus, resolve ions that may

56

be indistinguishable by MS alone, or to determine structural information like

57

rotationally averaged collision cross-sectional area (CCS). Moreover, IM-MS

58

offers the chance to obtain insights into the conformational dynamics of a

59

system, sharing unique means of characterizing flexibility and folding

60

changes.1,2

61

Quite a few publications deal with the power of IM-MS in positive ion mode for

62

structural characterization and the study of conformational dynamics of

63

proteins, the relationship between protein crystal structures and their CCS in

64

the gas phase. As nonnative protein conformations are rarely isolated in

65

solution, they are often stable in the gas phase in varying charge states.

66

Differences in the structures of nonnative conformations in the gas phase are

67

often large enough to allow different charge states and shapes enabling

68

separation because of differences in their mobilities through a gas.2-4

69

Recently, IM-MS has been recognized for having significant research/applied

70

industrial potential and comprises multi-/cross-disciplinary areas of science,

71

the applications and impact from decades of research are only now beginning

72

to be utilized for small molecule species.5 CCS was used as additional

73

identification point in pesticide analysis,6,7 IM-MS to investigate different

3



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conformers of 25-hydroxyvitamin D3,8 positional isomers of drug metabolites

75

and glucuronide positional isomers,9,10 isomeric carotenoids,11 regioisomers of

76

methylenedianiline,12 and protonation isomers.13,14 Interestingly, most of the

77

described applications also used the positive ion mode for small molecules.

78

Only few publications deal with the negative ion mode, e.g. the structural

79

characterization of the deprotonated leucine-enkephaline peptide anion or

80

glucuronide positional isomers of naringenin as well as β-estradiol.10,15

81

The genus Garcinia, familiy Guttiferae, exists in about 400 species in which

82

extracts and pure isolates causes different forms of bioactivity such as anti-

83

inflammatory,16 antimicrobial,17 anticancer,18 and antioxidant properties.19-21

84

Stem and root bark extracts of G. buchananii are traditionally used in Africa to

85

treat various conditions associated with HIV/AIDS such as herpes zoster,

86

cryptococcal meningitis, tuberculosis and chronic diarrhea, diabetes and

87

cardiovascular diseases as well as illnesses including diarrhea and pain.22-25

88

In recent years, G. buchananii has attracted interest as its stem bark extract

89

exhibit anti-diarrheal and antinociceptive as well as antioxidant effects.26-30

90

Activity-guided fractionation of stem and root bark as well as

91

phytochemical analysis of leaf extract of G. buchananii lead to the discovery

92

of

93

polyisoprenylated benzophenones,32,33 xanthones,32,33 the seco-xanthone 1,5-

94

dimethoxyajacareubin,32 the

95

glycosides,30,35 (3α8'') and (3α6'')-linked biflavonoids, mainly biflavanones

96

and -glycosides.30,32-38 We discovered that (2R,3S,2''R,3''R)-manniflavanone

97

(17, numbering refers to Table 1) is the major compound of G. buchananii

98

bark extract and also prominent in root and leaf extract and exhibits very

numerous

compounds

including

benzoyl

depsidone

4

glucuronosyl

glycerols,31

garcinisidone-G,32 flavanone-C-


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99

potent in vitro antioxidant activities.30,32,35 Biological tests revealed that 17

100

protects proliferating skeletal muscle cells against oxidative stress, stimulates

101

myotube formation and causes a relaxation of gut smooth muscle by

102

modulating calcium mobilization including the influx by means of L-type

103

calcium channels.39,40

104

To date, neither chemical nor bioactivity investigations have been performed

105

on extracts from branch bark, fruit rind, fruit flesh and seed of G. buchananii.

106

Therefore, this study had four aims: (i) to build a database under UNIFI

107

informatics platform from Waters Corporation consisting of retention time,

108

accurate m/z of the corresponding precursors, accurate m/z of the most

109

prominent fragment ions and CCS with 34 isolated pure reference compounds

110

from G. buchananii, (ii) to demonstrate the “proof of concept” using various so

111

far not investigated organs of G. buchananii, (iii) to demonstrate the potential

112

for future applications by applying the database to other Garcinia species (G.

113

mangostana, G. kola and G. cambogia), and (iv) to characterize deprotonated

114

polyphenol anions in the gas phase yielding in more than one CCS value of a

115

compound and, therefore, in a new characteristic compound parameter, the

116

drift time distribution intensity ratio.

117 118

MATERIALS AND METHODS

119

Chemicals. H2O for chromatographic separations was purified with a

120

Milli-Q Gradient A10 system (Millipore, Schwalbach, Germany), and solvents

121

used were of HPLC-MS-grade (Merck, Darmstadt, Germany). Deuterated

122

solvents were obtained from Euriso-Top (Saarbrücken, Germany).

5



123

Plant Material. Leaves, stem and root bark from G. buchananii trees in

124

their natural habitats in Masheshe, Karagwe, Tanzania. In addition, root bark,

125

stem bark, branch bark, leaves and fruit from G. buchananii were collected

126

from trees in their natural habitats in Kamagambo, Karagwe, Tanzania. Fruits

127

were peeled to obtain fruit rind, flesh and seeds. All samples were dried and

128

grounded to powder. A voucher specimen of the powders were deposited at

129

the University of Idaho Stillinger herbarium (voucher # 159918). Ethanolic G.

130

kola seed extract (origin South Africa) was obtained from PepsiCo (New York,

131

USA) and G. mangostana fruit and fruit juice (Hanoju Bio Mangosteen Saft

132

Premium 100%) were purchased in a local supermarket (Freising, Germany).

133

The Hanoju Bio Mangosteen juice was freeze-dried (90 ml) to yield 10.7 g

134

powder. The fruit was opened and separated into juice, fruit bark, flesh,

135

seeds, fruit shaft and sepal and freeze-dried, respectively. G. cambogia whole

136

fruit capsules (500 mg per capsule) as food supplement were purchased via

137

Amazon.

138

Extraction and Isolation. Extraction of G. buchananii seed powder and

139

MPLC separation was performed as described for root, leaf and bark

140

powder.30-33 14 fractions (M1-M14) were collected, concentrated under

141

reduced pressure and freeze-dried. 12 was isolated from fraction M5 and 13

142

from M6. Chromatography of MPLC fraction 5 was performed using a RP

143

column (10  250 mm, phenylhexyl, 5 μm; Phenomenex, Aschaffenburg,

144

Germany) as the stationary phase. The effluent (4.2 ml/min) was monitored at

145

270 nm and an isocratic gradient consisting of a mixture (53/47, v/v) of

146

aqueous HCO2H (0.1% in H2O) and MeOH was applied. Collected fractions

147

were concentrated under reduced pressure and freeze-dried twice, affording 6


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148

(2R,3S,2''R,3''R)-GB-2 7''-O-β-D-glucopyranosyl-6''''-malonic acid (12, Figure

149

1): colorless powder; UV (MeOH) λmax = 335, 288, 227, 206 nm; CD (MeOH,

150

0.66 mmol/L): λmax (ΔƐ) = 351 (+1.4), 309 (-3.2), 283 (+11.6), 236 (-5.8), 232 (-

151

4.9), 217 (-11.6), 203 (-0.2); HRESIMS m/z 821.1568 [M-H]- (calcd for

152

C39H33O20, 821.1565); 1H NMR (DMSO-d6, 27 °C, 500 MHz) data, see Table

153

1; 13C NMR (DMSO-d6, 27 °C, 125 MHz) data, see Table 2.

154

13 was obtained from M6 by semipreparative HPLC (4.2 ml/min, 270 nm)

155

using a ThermoHypersil ODS (10  250 mm, 5 μm; Kleinostheim, Germany)

156

column as the stationary phase and a mixture (62/38, v/v) of aqueous HCO2H

157

(0.1% in H2O) and MeOH, and the MeOH content was linearly increased to

158

41% within 20 min. Collected fractions were concentrated under reduced

159

pressure and freeze-dried twice, affording (2R,3S,2''R,3''R)-GB-1 7''-O-β-D-

160

glucopyranoside (13, Figure 1): colorless powder; UV (MeOH) λmax = 338,

161

289, 226 nm; CD (MeOH, 0.49 mmol/L): λmax (ΔƐ) = 369 (+2.0), 315 (-4.3),

162

287 (+9.1), 249 (-4.2), 243 (-4.0), 230 (-10.7), 226 (-10.3), 222 (+11.9), 204

163

(+0.1), 202 (-0.7); HRESIMS m/z 719.1625 [M-H]- (calcd for C26H31O16,

164

719.1612);

165

Information (SI) Table S1;

166

Table S2 (SI).

167

Ultra

1H

NMR (DMSO-d6, 27 °C, 500 MHz) data, see Supporting

Performance

13C

NMR (DMSO-d6, 27 °C, 125 MHz) data, see

Liquid

Chromatography

–

Electrospray

168

Ionisation-Ion Mobility-Time-of-Flight Mass Spectrometry (UPLC-ESI-IM-

169

TOF MS).

170

Plant material (G. buchananii and mangostana, 5 mg each) were suspended

171

in a mixture of acetonitrile and water (1:1, v/v, 1 ml), vortexed for 1 min,

172

ultrasonicated (10 min) and centrifuged (5000 rpm, 5 min). The obtained

7



173

extracts were membrane filtered and diluted to a final volume of 1 ml. Fruit

174

and fruit juice extracts were prepared in a tenfold higher concentration.

175

Ethanolic G. kola seed extract was prepared in a concentration of 2 mg/ml

176

(acetonitrile/water 1:1, v/v). G. cambogia whole fruit capsule extracts were

177

prepared as described above (1 and 10 mg/ml) and, additionally, a solid

178

phase extraction (Chromabond C18ec, 1g, 60 Å, 45 µm, Macherey-Nagel,

179

Düren, Germany) with whole fruit capsule (1.2 g) was performed. The SPE

180

column was activated, loaded with the sample, washed with 1% acetonitrile

181

and flushed with MeOH (70 ml), the MeOH fraction collected, evaporated and

182

dissolved in MeOH (4 ml).

183

Aliquots (8 µl) of the extracts were analyzed by means of UPLC-ESI-TWIMS-

184

TOF MS on a Waters Vion HDMS mass spectrometer (Waters, Manchester)

185

coupled to an Acquity i-class UPLC system (Waters, Milford) equipped with a

186

2 x 150 mm, 1.7 µm, BEH C18 column (Waters) consisting of a binary solvent

187

manager, sample manager and column oven. Operated with a flow rate of 0.4

188

ml/min at 45 °C, the following gradient was used for chromatography: starting

189

with a mixture (10/90, v/v) of aqueous HCO2H (0.1% in H2O) and MeCN

190

(0.1% HCO2H), the MeCN content was increased to 40% within 8 min, to 80%

191

within 1 min, to 90% within 2 min, to 100% within 1 min, kept constant for 1

192

min, decreased to 10% within 1 min and finally kept constant for 1 min at 10%.

193

Scan time for the HDMSe method was set to 1.0 sec. Analyses were

194

performed in ESI sensitivity mode using the following ion source parameters:

195

capillary voltage 2.3 kV in negative mode and 3.0 kV in positive mode, source

196

temperature 120 °C, desolvation temperature 550 °C, cone gas flow 50 l/h

197

and desolvation gas 900 l/h. Data processing was performed by using UNIFI

8


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Page 9 of 40


198

1.8 (Waters). All data were lock mass corrected on the pentapeptide leucine

199

enkephaline (Tyr-Gly-Gly-Phe-Leu, m/z 554.2615, [M-H]-) in a solution (50

200

pg/µl) of ACN/0.1% HCO2H (1/1, v/v). Scan time for the lock mass was set to

201

2.0 s with an interval of 0.5 min. Calibration of the Vion in the range from m/z

202

50 to 1200 was performed using a solution of the MajorMixTM (Waters). The

203

UPLC and Vion systems were operated with UNIFITM software (Waters).

204

Collision energy ramp for HDMSe was set from 20 to 30 eV. Further details of

205

the Vion IMS qTof instrument and processing and detection parameters for

206

application of the database are listed in the SI (Table S3).

207 208

RESULTS AND DISCUSSION

209

By means of UPLC-UV-ESI-TOF-MS we detected two biflavanone

210

glycosides as major constituents next to manniflavanone in G. buchananii

211

seed extract, therefore, compounds 12 and 13 were isolated and

212

characterized.

213

Compound 12 (Figure 1) was isolated as a colorless powder and

214

showed a pseudomolecular ion peak m/z 821.1568 [M-H]- (calcd. 821.1565) in

215

the HR-ESI-MS (Figure S1, SI), corresponding to the molecular formula of

216

C39H34O20. The UV spectrum was very similar to reported biflavanones30,32-38

217

and the loss of 44 as well 162 Da in the high-collision energy mass spectrum

218

(MSe) was representative for a decarboxylation and hexose unit which yielded

219

the elemental composition of GB-2 or buchananiflavanone. However, further

220

characteristic mass fragments of m/z 447, 429, 419, 403, 269, and 125

221

indicated a GB-2 aglycone (Figure S2, SI).38

222

The 1H NMR spectrum of compound 12 (Figure S4, SI) in DMSO-d6 at

223

27 °C showed sharp signals and the typical signal doubling as known from

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224

literature for biflavananones,30,32,33,35,38 indicating rotational conformers in a

225

ca. 3:1 ratio. The sets of aliphatic C-ring protons H-3 and H-2 as well as H-3''

226

and H-2'' coupling with each other were observed substantiating the GB-2

227

biflavananone aglycone structure. The coupling constants of J=12 Hz of these

228

protons verified their diaxial (trans) orientation. The COSY,

229

as well as 1H-13C HMBC NMR spectra (Figures S5-S8, SI) indicated the

230

presence of two carboxyl (δC 167.2, 168.1) and two carbonyl carbons (δC

231

195.5, 196.8, 197.9), 19 methine carbons, two methylene, and 14 further

232

carbons and were quite similar to that of (2R,3S,2''R,3''R)-GB-2 7''-O-β-D-

233

glucopyranoside.38 One methylene and two carboxyl groups could be

234

additionally detected which highlighted a malonic acid residue. The complete

235

assignments of 1H and

236

Tables 1 and 2. The fingerprint correlation in the 1H-13C HMBC spectrum

237

(Figure S8, SI) of both conformers between the proton H-3 and neighboring

238

carbon atom C-8'', C-8a'', and C-7'' demonstrated the intramolecular (38'')-

239

linkage of the two flavanone constituent units. The β-D-glucopyranosyl moiety

240

in both conformers was located at C-7'' as evidenced by the HMBC correlation

241

of the anomeric proton with C-7''. The couplings of the diastereotopic protons

242

H-6'''' to the carboxyl C-1''''' disclosed the linkage of the malonic acid unit to C-

243

6'''' of the β-D-glucopyranosyl moiety. The absolute configuration of 12 was

244

defined by comparison of its experimental and the published ECD curves of

245

(2R,3S,2''R,3''R)-manniflavanone 7‘‘-O-β-D-glucp and (2R,3S,2''R,3''R)-GB-2

246

7‘‘-O-β-D-glucp.38 The results showed that the ECD curve of 12 (Figure S9,

247

SI) was consistent with the ECD spectrum of the corresponding glycosides as

248

well as aglycones.30,35,38 On the basis of these data, compound 12 was

13C

13C, 1H-13C

HSQC

NMR of the two conformers at 27 °C are given in

10


Page 11 of 40


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assigned as (2R,3S,2''R,3''R)-GB-2 7''-O-β-D-glucopyranosyl-6''''-malonic acid,

250

a

251

biflavanone glucopyranosyl malonic acid derivative.

new

compound,

an

ent-naringenin-(C3C8'')-dihydroquercetin-linked

252

Compound 13 (Figure 1) was isolated as a colorless powder and

253

showed a pseudomolecular ion peak m/z 719.1613 [M-H]- (calcd. 719.1612) in

254

the HR-ESI-MS (Fig. S10, SI), corresponding to the molecular formula of

255

C26H32O16. 1 and 2 D NMR of the major conformer, CD and UV spectroscopy

256

as well as mass spectrometry (Figures S11-S17, SI) were in line to

257

(2R,3S,2''R,3''R)-GB

258

glucopyranoside which was earlier described in unripe fruits of Clusia

259

paralicula.41

1

as

well

as

(2R,3S,2''R,3''R)-GB-1

7''-O-β-D-

260

34 isolated and fully characterized reference compounds from G.

261

buchananii were analyzed by means of UPLC-ESI-TOF-MS on a VION IMS-

262

QTOF mass spectrometer in the negative ion mode to build a database under

263

UNIFI informatics platform consisting of retention time, accurate m/z of the

264

corresponding precursors, accurate m/z of the most prominent fragment ions

265

and CCS (Table 3). It is interesting to mention that by means of IM and the

266

resulting drift time the different compound classes could be clearly separated:

267

the three xanthones showed the lowest CCS with 143-174 Å2, followed by the

268

two benzoyl glucuronosyl glycerols as well as six flavanone-C/O-glycosides or

269

flavonol-C-glycoside with CCS of 179-185 Å2 or 199-206 Å2, respectively. The

270

11 biflavonoids were evaluated with 219-225 Å2, the four polyisoprenylated

271

benzophenones with 249-250 Å2 and the four biflavanone–glycosides with

272

256-263 Å2. Only four compounds do not belong to one of the six compound

273

classes, the seco-xanthone 1,5-dimethoxyajacareubin (27) with a CCS of

11



Page 12 of 40

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190.3 Å2, the depsidone garcinisidone-G (30) (183.2 Å2), the flavanone-

275

chromone biflavonoid preussianone (15) (194.1 Å2) and the diglycoside

276

apigenin-8-C-β-D-glcp-2''-O-L-rhap (9) (227.3 Å2) (Figure 2).

277

To check the stability of the CCS in a matrix sample, the intra- as well

278

as interday precision of the CCS of six selected compounds in a G.

279

buchananii bark extract was analyzed over a concentration range of three

280

magnitudes (Table 4). Additionally, the influence on the CCS area of different

281

solvents and formic acid ratio used for the chromatography, and therefore of

282

the ionization process, was investigated. In summary, the standard deviation

283

(SD) ranged between 0.1 and 0.9% and the average over 129 data points is

284

0.5%, which represents excellent precision of the CCS area. Consequently,

285

the CCS value is a compound specific parameter like m/z or fragments and

286

independent from the chromatographic conditions which could also be

287

combined with direct MS infusions. This is a very big advantage compared to

288

the retention time of a compound which changes with other column chemistry,

289

length, column age, flow rate, temperature as well as solvents and buffers.

290

In a next step, the established database was applied on different

291

organs of G. buchananii, including stem, branch, fruit and root bark, as well as

292

leaves, seeds and fruit flesh and the peak targeting is based on four fulfilled

293

criteria (Table 3): retention time (±0.12 min), accurate m/z of the

294

corresponding precursors (± 10 ppm), accurate m/z of the most prominent

295

fragment ions (± 10 mDa) and CCS (± 1.2 %). To the best of our knowledge,

296

branch and fruit bark as well as seeds and fruit flesh have never been

297

investigated so far. 22 compounds were detected in stem bark, mainly

298

biflavanoid

glycosides

and

their

corresponding

12


aglycones,

flavonoid

Page 13 of 40


299

glycosides, polyisoprenylated benzophenones as well as the xanthone

300

jacareubin (29) (Table S4, SI). In root and branch bark the same number and

301

a very similar composition could be verified like in stem bark, with just minor

302

differences in 15 as well as ulmoside A (5), helicioside A (7) and isoprenyl

303

tetra hydroxyxanthone (28) (Table S5-S6, SI). In leaves, 25 compounds could

304

be identified, similar components to the barks mentioned so far, but no

305

xanthones instead 30 as well as the two benzoyl glucuronosyl glycerols (3, 6)

306

(Table S7, SI). The analysis of the seeds showed 16 identified compounds

307

and to be a rich source of isogarcinol (34) and mainly biflavanoid glycosides

308

and their corresponding aglycones as well as flavonoid glycosides (Table S8,

309

SI). It is interesting to mention that among others the edible part of G.

310

buchananii fruit contains the most components, namely 26. All compound

311

classes

312

benzophenones and the recently in leaves identified 1-O-4-hydroxybenzoyl-

313

3-O-α-D-glucuronosylglycerine (3). The fruit rind extract revealed 25

314

compounds with similar composition to the fruit in which garcicowin C (32),

315

garcinol (33) and 15 could not be observed, but found 27 and quercetin-6-C-

316

β-D-glcp (1).

are

represented,

highlighting

several

polyisoprenylated

317

In summary, the extracts of different organs of G. buchananii tree

318

principally showed a similar chemical composition pattern, as some

319

compounds are ubiquitous, like e.g. GB-1, GB-2, manniflavanone, -O-

320

glycoside, buchananiflavanone, isogarcinol but there were also clear

321

differences. For example: 3, 9, 10, 23 and 25 are markers for leaves,31,33 fruit

322

and fruit rind. Unique organ markers are euxanthone (26) for fruit, 6 and 30 for

323

leaves, 12 for seeds, 27 for fruit bark and 28 for branch bark. For the proof of

13



324

principle, the database constructed with compounds isolated from seeds,

325

leaves, stem and root bark was applied on the corresponding extracts and on

326

additional organs not investigated so far, namely, branch bark, fruit rind and

327

fruit extract and yielded fast and very reliable results based on four fulfilled

328

criteria.

329

To strengthen the benefits of the constructed database, we applied it to

330

other Garcinia species, namely, G. kola (seeds), G. mangostana (juice, pulp,

331

seeds, sepal, fruit shaft, exo- and mesocarp) and G. cambogia (whole fruit

332

capsules). 14 compounds could be identified in an ethanolic extract of G. kola

333

seeds (Table S11, SI), whereas 7, (2R,3S,2‘‘S)-buchananiflavanon (11), 9, 26,

334

32-34 could be detected for the first time. The application of the database on

335

different organs of G. mangostana revealed six different compounds, thereof

336

five (3, 4, 6, 10 and 29) were identified for the first time in this species (Table

337

S12, SI). In contrast, the analysis of the whole fruit capsules of G. cambogia,

338

also additionally enriched via SPE, did not show any hit. This is surprising as

339

at least 33 along with other polyisoprenylated benzophenones as well as

340

xanthones have already been described in G. cambogia fruits.42

341

Positive ESI in combination with TWIM-MS could resolve or indicate

342

protomers of small molecules or different confomers or proteins,2,13,14 in

343

contrast, in the negative ESI mode such investigations are rarely found, like

344

deprotomers of ortho, meta, and para hydroxybenzoic acid and a

345

deprotonated porphyrin derivative.43,44

346

By means of NMR spectroscopy we observed rotational conformers in

347

solution for compounds 8 and 11-25,30,32,33,35,38 and, therefore, had a closer

348

look on the TWIM-MS data of these compounds. Exemplified for 17 (Figure

14


Page 14 of 40

Page 15 of 40


349

3A), two different drift time species could be detected, which showing identical

350

retention time, accurate mass and fragments, but a CCS of 224.1 and 309.1

351

Å2 (Table 3) and an intensity ratio of 95.3/4.7%. To clarify if the detected drift

352

time species could be attributed to either deprotomers, rotational isomers or in

353

general to gas phase conformers, the nine fold methylated analogue of 17

354

was analyzed, only highlighting one drift time species and proposing that the

355

hydrogen bond stabilized rotational isomers 17a and 17b were transferred

356

from solution to gas phase (Figure 3B).

357

positive ion mode revealed very similar CCS values and, especially, a drift

358

time intensity distribution of 94.7/5.3%, which further strengthen our

359

suggestion. We have exemplary checked the intra- and interdaily stability of

360

the intensity of the drift time distribution of the manniflavanone (17)

361

conformers under our conditions as well as different solvents listed in Table 5.

362

Intraday a ratio of 95.2/4.8%±0.1% RSD, interday of 96.0/4.0%±0.2%RSD

363

and with different solvents and acid concentration of 96.4/3.6%±0.5% RSD

364

was determined. The mean over all (21) analyses is 96.0/4.0±0.6%RSD which

365

seems to be quite robust. Other compounds showed comparable data. The

366

data for the diverse drift time intensity distributions is summarized in Table 6.

367

With the exception of 8 and 12, basically, the denser drift time species is

368

dominating, at least 72% for 2, normally >80-90%. It is very interesting to

369

mention, that vice versa for the corresponding glycosides 8 and 12 the higher

370

CCS is dominating. This observation was further strengthened by the

371

glycosides 11 (data not shown) and 13 which showed the same shift (Figure

372

4). We propose to use all CCS values of one compound, and also the intensity

373

ratio of the conformers as a new characteristic compound parameter next to

15

UPLC-ESI-TWIMS of 17 in the



374

m/z, fragments, Rt and CCS in compound identification/screening/database

375

applications to reduce false positives and strengthen the identification. By

376

plotting the mass vs. the CCS values of the major rotational conformers (in

377

case of two or more drift time species) of all compounds the strong linear

378

correlation between molecular weight and CCS can be depicted which is in

379

line with literature (Figure 2).5,7 By trend, increasing molecular weight reflects

380

in increasing CCS whereas to keep in mind that this plot was performed with

381

the major conformer. In TWIMS, bigger ions roll over the wave, whereas

382

smaller ions “surfing” on the traveling wave exciting the gas-filled mobility cell

383

earlier. By comparing manniflavanone (17) with its 7’’-O-glucoside (8), and

384

therefore 162 Da heavier, the CCS of the major conformers (224.3  261.7

385

Å2) seems to follow the general trend with increasing molecular weight

386

(Figure 5). But if we directly compare the drift time distributions of the first and

387

the second conformers the CCS values are decreasing (309.0  261.7 and

388

224.3  212.6 Å2) with increasing m/z, respectively, or more precisely, by

389

glycosylation (Figure 5). The same results could be determined for 21 and 18

390

as well as their corresponding glycosides 11 and 13 and even more for 12, the

391

glucopyranosyl malonic acid derivative of GB-2 (Table 5). Further studies on

392

the CCS values of the isomers of manniflavanone 14, 17 and 19 revealed that

393

the major drift time species of these three compounds are sharing the same

394

CCS value of 224 Å2 although the discrimination is possible via the minor

395

conformer (no minor conformer for 14, 326.2 for 19 and 309.0 Å2 for 17) and

396

therefore enables the identification of the isomers in plant extracts by means

397

of direct infusion experiments without column chromatography (Table 3 and

398

5). Another example for the influence of the stereochemistry on the CCS

16


Page 16 of 40

Page 17 of 40


399

value is the comparison of (2R,3R)-taxifolin-6-C-β-D-glucopyranoside (2) and

400

its diastereomer ulmoside A (5), differing in the configuration of the agycone.

401

Interestingly, both stereoisomers showed two drift time species, probably

402

gaseous conformers, whereas the CCS of the major conformer of 2 and of the

403

minor conformer of 5 is bigger and the values differed in 4 Å2, each. Also the

404

intensity ratios of the two drift time species differed clearly. Moreover, 2

405

highlighted the possibility of a third drift time species (Figure 6). Again, the

406

identification of 2 or 5 in plant extracts would be possible by means of direct

407

infusion experiments without column chromatography and TWIMS with the

408

different CCS values and intensity ratios of the drift time species (Table 3 and

409

5).

410

Although literature and our own studies showed a high correlation between

411

m/z and CCS trusting on m/z alone to predict CCS is not adequate as many

412

ions have the same m/z but different 3D structure as demonstrated for the

413

stereoisomers (2 and 5), and, especially glycosylation increases the molecular

414

weight by 162 Da but yielded in a more compact structure and smaller CCS

415

(8, 11-13). We demonstrated that the CCS value is a robust and precise

416

compound parameter and to use all CCS values of one compound, and

417

beyond that propose the intensity ratio of the conformers as a new

418

characteristic compound parameter next to m/z, fragments, Rt and CCS. The

419

constructed database could be used as a fast, robust and reliable

420

dereplication tool and the determined CCS values could be used in public,

421

growing databases.

422 423 424

ACKNOWLEDGEMENT

17



425 426

We thank PepsiCo (New York, USA) for providing the G. kola seed extract.

427

ASSOCIATED CONTENT

428 429 430

Supporting Information

431

UPLC-UV-ESI-TOF MS, MSe, CD, 1- and 2D NMR spectra of compound 12

432

and 13, and Tables summarizing all COSY, HSQC and HMBC couplings of

433

13, detailed instrument parameters of the Vion IMS qTOF and processing

434

options of the database as well as compound identification in the different

435

plant organs of G. buchananii and G. mangostana and G. kola seed extract

436

using the in house database. This information is available free of charge via

437

the Internet at http: //pubs.acs.org.

438

Notes

439

The authors declare no competing financial interest.

440 441

REFERENCES

442 443

(1)

Lanucara, F.; Holman, S. W.; Gray, C. J.; Eyers, C. E. The power of ion

444

mobility-mass spectrometry for structural characterization and the study

445

of conformational dynamics. Nat. Chem. 2014, 6, 281-94.

446

(2)

Shvartsburg, A. A.; Smith, R. D., Separation of Protein Conformers by

447

Differential Ion Mobility in Hydrogen-Rich Gases. Anal. Chem. 2013,

448

85, 6967-6973.

449

(3)

Bohrer, B. C.; Merenbloom, S. I.; Koeniger, S. L.; Hilderbrand, A. E.;

450

Clemmer, D. E. Biomolecule analysis by ion mobility spectrometry.

451

Annu. Rev. Anal. Chem. (Palo Alto Calif) 2008, 1, 293-327.

18


Page 18 of 40

Page 19 of 40

452


(4)

Jurneczko, E.; Barran, P. E. How useful is ion mobility mass

453

spectrometry for structural biology? The relationship between protein

454

crystal structures and their collision cross sections in the gas phase.

455

Analyst 2011, 136, 20.

456

(5)

Lapthorn, C.; Pullen, F.; Chowdhry, B. Z. Ion mobility spectrometry-

457

mass spectrometry (IMS-MS) of small molecules: separating and

458

assigning structures to ions. Mass. Spectrom. Rev. 2013, 32, 43-71.

459

(6)

Bijlsma, L.; Bade, R.; Celma, A.; Mullin, L.; Cleland, G.; Stead, S.;

460

Hernandez, F.; Sancho, J. V. Prediction of collision cross-section

461

values for small molecules: application to pesticide residue analysis.

462

Anal. Chem. 2017, 89, 6583-6589.

463

(7)

Regueiro, J.; Negreira, N.; Berntssen, M. H. Ion-mobility-derived

464

collision cross section as an additional identification point for

465

multiresidue screening of pesticides in fish feed. Anal. Chem. 2016, 88,

466

11169-11177.

467

(8)

Chouinard, C. D.; Cruzeiro, V. W. D.; Beekman, C. R.; Roitberg, A. E.;

468

Yost, R. A. Investigating differences in gas-phase conformations of 25-

469

hydroxyvitamin

470

spectrometry and theoretical modeling. J. Am. Soc. Mass. Spectrom.

471

2017, 28, 1497-1505.

472

(9)

D3

sodiated

epimers

using

Ion

mobility-mass

Cuyckens, F.; Wassvik, C.; Mortishire-Smith, R. J.; Tresadern, G.;

473

Campuzano, I.; Claereboudt, J. Product ion mobility as a promising tool

474

for assignment of positional isomers of drug metabolites. Rapid

475

Commun. Mass Spectrom. 2011, 25, 3497-503.

19



476

(10)

Page 20 of 40

Reading, E.; Munoz-Muriedas, J.; Roberts, A. D.; Dear, G. J.;

477

Robinson, C. V.; Beaumont, C. Elucidation of drug metabolite structural

478

isomers using molecular modeling coupled with ion mobility mass

479

spectrometry. Anal. Chem. 2016, 88, 2273-2280.

480

(11)

Dong, L.; Shion, H.; Davis, R. G.; Terry-Penak, B.; Castro-Perez, J.;

481

van Breemen, R. B. Collision cross-section determination and tandem

482

mass spectrometric analysis of isomeric carotenoids using electrospray

483

ion mobility time-of-flight mass spectrometry. Anal. Chem. 2010, 82,

484

9014-21.

485

(12)

Forsythe, J. G.; Stow, S. M.; Nefzger, H.; Kwiecien, N. W.; May, J. C.;

486

McLean, J. A.; Hercules, D. M. Structural characterization of

487

methylenedianiline regioisomers by Ion mobility-mass spectrometry,

488

tandem

489

Electrospray spectra of 2-ring isomers. Anal. Chem. 2014, 86, 4362-

490

4370.

491

(13)

mass

spectrometry,

and

computational

strategies:

I.

Boschmans, J.; Jacobs, S.; Williams, J. P.; Palmer, M.; Richardson, K.;

492

Giles, K.; Lapthorn, C.; Herrebout, W. A.; Lemiere, F.; Sobott, F.

493

Combining density functional theory (DFT) and collision cross-section

494

(CCS) calculations to analyze the gas-phase behaviour of small

495

molecules and their protonation site isomers. Analyst 2016, 141, 4044-

496

54.

497

(14)

Warnke, S.; Seo, J.; Boschmans, J.; Sobott, F.; Scrivens, J. H.;

498

Bleiholder, C.; Bowers, M. T.; Gewinner, S.; Schollkopf, W.; Pagel, K.;

499

von Helden, G. Protomers of benzocaine: solvent and permittivity

500

dependence. J. Am. Chem. Soc. 2015, 137, 4236-42.

20


Page 21 of 40

501


(15)

Schinle, F.; Jacob, C. R.; Wolk, A. B.; Greisch, J. F.; Vonderach, M.;

502

Weis, P.; Hampe, O.; Johnson, M. A.; Kappes, M. M. Ion mobility

503

spectrometry, infrared dissociation spectroscopy, and ab initio

504

computations toward structural characterization of the deprotonated

505

leucine-enkephalin peptide anion in the gas phase. J. Phys. Chem. A

506

2014, 118, 8453-63.

507

(16) Obolskiy, D.; Pischel, I.; Siriwatanametanon, N.; Heinrich, M. Garcinia

508

mangostana

L.:

A

phytochemical

509

Phytother. Res. 2009, 23, 1047-1065.

and

pharmacological

review.

510

(17) Naldoni, F. J.; Claudino, A. L. R.; Cruz, J. W.; Chavasco, J. K.; Faria, E.;

511

Silva, P. M.; Veloso, M. P.; Dos Santos, M. H. Antimicrobial activity of

512

benzophenones and extracts from the fruits of Garcinia brasiliensis. J.

513

Med. Food 2009, 12, 403-407.

514

(18) Han, Q. B.; Yang, N. Y.; Tian, H. L.; Qiao, C. F.; Song, J. Z.; Chang, D.

515

C.; Chen, S. L.; Luo, K. Q.; Xu, H. X. Xanthones with growth inhibition

516

against HeLa cells from Garcinia xipshuanbannaensis. Phytochem.

517

2008, 69, 2187-2192.

518

(19) Joseph, G. S.; Jayaprakasha, G. K.; Selvi, A. T.; Jena, B. S.; Sakariah,

519

K. K. Antiaflatoxigenic and antioxidant activities of Garcinia extracts. Int.

520

J. Food Microbiol. 2005, 101, 153-160.

521 522

(20) Okoko, T. In vitro antioxidant and free radical scavenging activities of Garcinia kola seeds. Food Chem. Toxicol. 2009, 47, 2620-2623.

523

(21) Kolodziejczyk, J.; Masullo, M.; Olas, B.; Piacente, S.; Wachowicz, B.

524

Effects of garcinol and guttiferone K isolated from Garcinia cambogia on

21



525

oxidative/nitrative modifications in blood platelets and plasma. Platelets

526

2009, 20, 487-492.

527

(22) Kisangau, D. P.; Lyaruu, H. V.; Hosea, K. M.; Joseph, C. C. Use of

528

traditional medicines in the management of HIV/AIDS opportunistic

529

infections in Tanzania: a case in the Bukoba rural district. J. Ethnobiol.

530

Ethnomed. 2007, 3, 29-37.

531

(23) Chinsembu, K. C., Hedimbi, M. An ethnobotanical survey of plants used

532

to manage HIV/AIDS opportunistic infections in Katima Mulilo, Caprivi

533

region, Namibia. J. Ethnobiol. Ethnomed. 2010, 6, 25.

534

(24) De Wet, H.; Nkwanyana, M. N.; van Vuuren, S. F. Medicinal plants used

535

for the treatment of diarrhoea in northern Maputaland, KwaZulu-Natal

536

Province, South Africa. J. Ethnopharmacol. 2010, 130, 284–289.

537

(25) Okullo, J. B. L.; Omujal, F.; Bigirimana, C.; Isubikalu, P.; Malinga, M.;

538

Bizuru, E.; Namutebi, A.; Obaa, B. B.; Agea J. G. Ethno-medicinal uses

539

of selected indigenous fruit trees from the lake Victoria basin districts in

540

Uganda. 2014, 2, 78–88.

541

(26) Balemba, O. B.; Bhattarai, Y.; Stenkamp-Strahm, C.; Lesakit, M. S. B.;

542

Mawe, G. M. The traditional anti-diarrheal remedy, Garcinia buchananii

543

stem bark extract, inhibits propulsive motility and fast synaptic potentials

544

in the guinea pig distal colon. Neurogastroent. Motil. 2010, 22, 1332-

545

1339.

546

(27) Boakye, P. A.; Brierley, S. M.; Pasilis, S. P.; Balemba, O. B. Garcinia

547

buchananii bark extract is an effective anti-diarrheal remedy for lactose-

548

induced diarrhea. J. Ethnopharmacol. 2012, 142, 539-547.

22


Page 22 of 40

Page 23 of 40


549

(28) Boakye, P. A.; Stenkamp-Strahm, C.; Bhattarai, Y.; Hechman, M. D.;

550

Brierley, S. M.; Pasilis, S. P.; Balemba, O. B. 5-HT3 and 5-HT4 receptors

551

contribute to the anti-motility effects of Garcinia buchananii bark extract

552

in the guinea-pig distal colon. Neurogastroent. Motil. 2012, 24, e27-40.

553

(29) Castro, J.; Balemba, O. B.; Blackshaw, L. A.; Brierley, S. M. Garcinia

554

buchananii bark extract inhibits nociceptors, with greater efficacy during

555

inflammation. Gastroenterology 2011, 140, Supplement 1, Page: S-866.

556

(30) Stark, T. D.; Matsutomo, T.; Lösch, S.; Boakye, P. A.; Balemba, O. B.;

557

Pasilis, S. P.; Hofmann, T. Isolation and structure elucidation of highly

558

antioxidative 3,8''-linked biflavanones and flavanone-C-glycosides from

559

Garcinia buchananii bark. J. Agric. Food Chem. 2012, 60, 2053-2062.

560

(31) Stark, T. D.; Lösch, S.; Balemba, O. B.; Hofmann, T. Two new benzoyl

561

glucuronosyl glycerols from the leaves of Garcinia buchananii.

562

Phytochem. Lett. 2017, 19, 187-190.

563

(32) Stark, T. D.; Salger, M.; Frank, O.; Balemba, O. B.; Wakamatsu, J.;

564

Hofmann, T. Antioxidative compounds from Garcinia buchananii stem

565

bark. J. Nat. Prod. 2015, 78, 234-240.

566

(33) Stark, T. D.; Lösch, S.; Wakamatsu, J.; Balemba, O. B.; Frank, O.;

567

Hofmann, T. UPLC-ESI-TOF MS based metabolite profiling of the

568

antioxidative food supplement Garcinia buchananii. J. Agric. Food

569

Chem. 2015, 63, 7169-7179.

570

(34) Jackson, B.; Locksley, H. D.; Scheinmann F. The structure of the “GB”

571

Biflavanones from Garcinia buchananii Baker and G. eugeniifolia Wall.

572

Chem. Commun. 1968, 18, 1125-1127.

23



Page 24 of 40

573

(35) Stark, T. D.; Germann, D.; Balemba, O. B.; Wakamatsu, J.; Hofmann, T.

574

New highly in vitro antioxidative 3,8''-linked biflav(an)ones and

575

flavanone-C-glycosides from Garcinia buchananii stem bark. J. Agric.

576

Food Chem. 2013, 61, 12572-12581.

577

(36) Jackson, B; Locksley, H. D.; Scheinmann F. Wolstenholme, W. A. The

578

isolation of a new series of biflavanones from the heartwood of Garcinia

579

buchananii. Tetrahedron Lett. 1967, 9, 787-792.

580

(37) Jackson, B.; Locksley, H. D.; Scheinmann, F.; Wolstenholme, W. A.

581

Extractives from Guttiferae. Part XXII. The isolation and structure of four

582

novel biflavanones from the heartwoods of Garcinia buchananii Baker

583

and G. eugeniifolia Wall. J. Chem. Soc. 1971, 22, 3791-3804.

584

(38) Stark, T. D.; Lösch, S.; Salger, M.; Balemba, O. B.; Wakamatsu, J.;

585

Frank, O.; Hofmann, T. A strategy to counteract heat-induced damage in

586

NMR

587

biflavanone-7''-O-β-D-glucopyranosides from Garcinia buchananii root.

588

Magn. Res. Chem. 2015, 53, 813-820.

experiments

to

characterize

biflavanones

and

two

new

589

(39) Balemba, O. B.; Stark, T. D.; Lösch, S.; Patterson, S.; McMillan, J. S.;

590

Mawe, G. M.; Hofmann, T. (2R,3S,2''R,3''R)-manniflavanone, a new

591

gastrointestinal smooth muscle L-type calcium channel inhibitor, which

592

underlies the spasmolytic properties of Garcinia buchananii stem bark

593

extract. J. Smooth Muscle Res. 2014, 50, 48-65.

594

(40) Acharya, S.; Stark, T. D.; Oh, S., Jeon, S.; Pak, S.; Kim, M.; Matsutomo,

595

T.; Hur, J.; Hofmann, T; Hill, R. A.; Balemba, O. B. (2R,3S,2’’R,3’’R)-

596

Manniflavanone protects proliferating skeletal muscle cells against

24


Page 25 of 40


597

oxidative stress and stimulates myotube formation. J. Agric. Food Chem.

598

2017, 65, 3636–3646.

599

(41) Oliveira, R. F.; Camara, C. A.; de Agra, M. F.; Silva, T. M. Biflavonoids

600

from the unripe fruits of Clusia paralicola and their antioxidant activity.

601

Nat. Prod. Commun. 2012, 7, 1597-1600.

602

(42)

Masullo, M.; Bassarello, C.; Suzuki, H.; Pizza, C.; Piacente, S.

603

Polyisoprenylated benzophenones and an unusual polyisoprenylated

604

tetracyclic xanthone from the fruits of Garcinia cambogia. J. Agric.

605

Food Chem. 2008, 56, 5205-5210.

606

(43)

Lalli, P. M.; Iglesias, B. A.; Toma, H. E.; de Sa, G. F.; Daroda, R. J.;

607

Silva Filho, J. C.; Szulejko, J. E.; Araki, K.; Eberlin, M. N. Protomers:

608

formation, separation and characterization via travelling wave ion

609

mobility mass spectrometry. J. Mass. Spectrom. 2012, 47, 712-719.

610

(44)

Galaverna, R. S.; Bataglion, G. A.; Heerdt, G.; de Sa, G.F.; Daroda, R.;

611

Cunha, V. S.; Morgon, N. H.; Eberlin, M. N. Are benzoic acids always

612

more acidic than phenols? The case of ortho-, meta-, and para-

613

hydroxybenzoic acids. Eur. J. Org. Chem. 2015, 2189-2196.

614

25



Table 1. 1H NMR data of the major and minor conformers of 12 at 27 ℃ (DMSO-d6, 500 MHz) δH in ppm (multiplicity, J in Hz) H no. Flavanone-unit (I) H no. Flavanone-unit (II) aA bB aA bB I-2 5.48 (d, 12.1) 5.71 (d, 12.1) II-2‘‘ 4.88 (d, 11.2) 4.91 (d, 11.9) I-3 5.09 (d, 12.1) 4.48 (d, 12.1) II-3‘‘ 4.25 (d, 11.2) 3.99 (d, 11.9) I-6 5.89 (d, 2.1) 5.84 (d, 1.6) II-6‘‘ 6.22 (s) 6.40 (s) I-8 5.77 (d, 2.1) 5.82 (d, 1.7) II-2‘‘‘ 6.81 (d, 2.0) 6.85 (d, 1.5) e I-2‘ 7.23 (d, 8.2) 7.09 (d, 8.3) II-5‘‘‘ 6.65 (d, 8.2) e I-3‘ 6.61 (d, 8.2) 6.74 (d, 8.3) II-6‘‘‘ 6.68 (dd, 2.0, 8.3) I-5‘ 6.61 (d, 8.2) 6.74 (d, 8.3) I-6‘ 7.23 (d, 8.2) 7.09 (d, 8.3) I-5-OH 12.08 (s) 11.97 (s) δH in ppm (multiplicity, J in Hz) H no. Glucopyranoside-unit H no. Malonic acid-unit aA bB aA bB d 1‘‘‘‘ 4.73 (d, 7.0) 5.02 (d, 7.8) 2‘‘‘‘‘ 3.34 (s) 3.35 2‘‘‘‘ 3.33c 3.12c 3‘‘‘‘ 3.33c 3.28c c 4‘‘‘‘ 3.17 3.13c 5‘‘‘‘ 3.67 (m) 3.67d e 6‘‘‘‘α 4.06 (dd, 7.4, 11.9) e 6‘‘‘‘β 4.37 (d, 10.9) Sets A and B are respectively in an intensity ratio of (~1:0.3) in 12. Additional OH signals as brs for 4', 3''' and 4''' at 11.71, 8.96, 5.75 and 4.70 ppm. aA represents major conformer of 12 at 27 ℃, bB represents minor conformer of 12 at 27 ℃, coverlapped with H O peak, assigned with COSY and HSQC 2 experiments. doverlapped with major signal, assigned with HSQC experiments. e not assignable, probably overlapped with signals of major conformer.

26


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Table 2. 13C NMR data of the major and minor conformers of 12 at 27 ℃ (DMSO-d6, 125 MHz) δC in ppm (C-Type) C no. Flavanone-unit (I) C no. Flavanone-unit (II) aA bB aA bB I-2 82.0 (CH) 81.3 (CH) II-2‘‘ 83.1 (CH) 82.8 (CH) I-3 46.7 (CH) 47.6 (CH) II-3‘‘ 72.1 (CH) 72.2 (CH) I-4 196.8 (C) 195.5 (C) II-4’’ 197.9 (C) 197.9 (C) I-4a 101.1 (C)c 101.4 (C) II-4a’’ 101.9 (C) 101.4 (C)c I-5 163.6 (C) 163.7 (C) II-5’’ 161.9 (C) 162.2 (C) I-6 95.9 (CH) 96.0 (CH) II-6’’ 96.2 (CH) 96.2 (CH) I-7 166.3 (C) 166.2 (C) II-7’’ 162.9 (C) 162.3 (C) I-8 94.8 (CH) 94.7 (CH) II-8’’ 102.4 (C) 102.5 (C) I-8a 162.8 (C) 162.7 (C) II-8a‘‘ 158.9 (C) 159.4 (C) I-1‘ 127.1 (C) 127.9 (C) II-1‘‘‘ 128.0 (C) 127.9 (C) d I-2‘ 129.8 (CH) 128.8 (CH) II-2‘‘‘ 115.1 (CH) 115.7 (CH)e I-3‘ 115.0 (CH) 114.8 (CH) II-3‘‘‘ 144.8 (C) 145.0 (C) I-4‘ 157.7 (C) 157.7 (C) II-4‘‘‘ 145.6 (C) 145.1 (C) I-5‘ 115.0 (CH) 114.8 (CH) II-5‘‘‘ 115.2 (CH)d 115.2 (CH)e I-6‘ 129.8 (CH) 128.8 (CH) II-6‘‘ 117.8 (CH) 119.0 (CH) δC in ppm (C-Type) C no. Glucopyranoside-unit C no. Malonic acid-unit aA bB aA bB f 1‘‘‘‘ 99.9 (CH) 99.7 (CH) 1‘‘‘‘‘ 167.2 (C) 2‘‘‘‘ 72.9 (CH) 73.5 (CH) 2‘‘‘‘‘ 41.9 (CH2) 41.6 (CH2) f 3‘‘‘‘ 75.9 (CH) 76.9 (CH) 3‘‘‘‘‘ 168.1 (C) 4‘‘‘‘ 69.9 (CH) 70.2 (CH) 5‘‘‘‘ 73.8 (CH) 74.0 (CH) f 6‘‘‘‘ 64.0 (CH2) *C-type was deduced from HSQC in combination with 1H experiment. aA represents major conformer at 27 ℃, bB represents minor conformer at 27 ℃, c,d,e

27



interchangeable, fnot observable, probably overlapped

28


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Table 3. The database under UNIFI informatics platform consisting of retention time, accurate m/z of the corresponding precursors,

accurate m/z of the most prominent fragment ions and CCS of 34 isolated reference compounds of G. buchananii. compound

Quercetin-6-C-β-D-glcp (2R,3R)-Taxifolin-6-C-β-D-glcp (2R)-1-O-4-hydroxybenzoyl-3O-α-D-glucuronosyl-glycerol (2R,3R)-Aromadendrin-6-C-β-D-glcp Ulmoside A ((2S,3S)-taxifolin-6-C-β-Dglcp) (2S)-1-O-4-hydroxy-3-methoxybenzoyl-3O-α-D-glucuronosyl-glycerol Helicioside A (2R,3S,2''R,3''R)-Manniflavanone-7''-O-βD-glcp Apigenin-8-C-β-D-glcp-2''-O-L-rhap Vitexin (2R,3S,2''R,3''R)-GB-2 7''-O-β-D-glycp (2R,3S,2''R,3''R)-GB-2 7''-O- β-D-glcp-6''''malonic acid (2R,3S,2''R,3''R)-GB-1 7''-O-β-D-glycp (2R,3S,2''S,3''S)-Manniflavanone (2''R,3''R)-Preussianone (2R,3S)-Buchananiflavonol

nr

expected RT (min)

1

1.46

expected neutral mass (Da) 464.0955

2 3

1.47 1.84

466.1111 388.1006

4 5

1.86 1.86

450.1162 466.1111

6

1.95

418.1111

7 8

2.62 3.24

450.1162 752.1589

9 10 11 12

3.48 3.59 3.62 4.07

578.1636 432.1056 736.1639 822.1643

13 14 15

4.38 4.67 5.31

720.1690 590.1060 480.0693

16

5.10

588.0904

29

expected fragment (m/z)

adduct

observed CCS (Å2)*

151.0037, 343.0459, 345.0616, 367.0459, 373.0565 167.0350, 345.0616, 375.0722 137.0244, 211.0612, 249.0616, 267.0722 301.0718, 329.0667, 359.0772 167.0350, 301.0718, 329.0667, 331.0823, 359.0772 108.0217,152.0115

-H

206.4

-H -H

198.6 177.8

-H -H

205.7 194.8

-H

185.2

259.0612, 269.0455 151.0037, 437.0878, 445.0565, 463.0671 293.0455, 311.0561, 413.0878 283.0612, 311.0561, 341.0667 403.0823, 429.0616, 447.0722, 447.0722, 429.0616, 777.1672

-H -H

205.5 263.3

-H -H -H -H

227.3 196.4 259.6 256.9

431.0772, 557.1089, 579.0933 435.0722, 445.0565, 463.0671 149.0244, 217.0142, 341.0667, 461.0514, 311.0561, 435.0722, 437.0878, 445.0565, 461.0514, 463.0671

-H -H -H

256.4 224.3 194.1

-H

218.8



(2R,3S,2''R,3''R)-Manniflavanone (2R,3S,2''R,3''R)-Manniflavanone, conformer (2R,3S,2''R,3''R)-GB-2 (2R,3S,2''R,3''R)-Isomanniflavanone (2R,3S,2''S)-Buchananiflavanone (2R,3S,2''R,3''R)-GB-1 (2R,3S,2´´S)-GB-2a (2R,3S)-Morelloflavone (2R,3S,2''S)-GB-1a (2R,3S)-Volkensiflavone Euxanthone 1,5-Dimethyoxyajacureubin 2-Isoprenyl-1,3,5,6-tetrahydroxyxanthone Jacareubin Garcinisidone G Paucinone C Garcicowin C Garcinol Isogarcinol

17a 17b

5.10 5.10

590.1060 590.1060

18 19

5.77 6.19

574.1111 590.1060

20

6.45

574.1111

21

6.51

558.1162

22 23 24 25 26 27 28 29

7.09 7.20 8.02 8.09 9.39 9.59 9.77 9.85

558.1162 556.1006 542.1213 540.1056 228.0423 356.1260 328.0947 326.0790

30 31 32 33 34

9.90 10.30 11.30 11.64 12.69

358.1053 634.3506 600.3451 602.3607 602.3607

285.0405, 435.0722, 463.0671 435.0722, 437.0878, 463.0671, 479.0620 296.0326, 419.0772, 447.0722 285.0405, 435.0722, 463.0671, 445.0565 285.0405, 311.0197, 421.0929, 447.0722 269.0455, 296.0326, 403.0823, 431.0772 269.0455, 295.0248, 431.0772 401.0667, 403.0823, 429.0616 295.0248, 389.1031, 415.0823 151.0037, 387.0874, 413.0667 210.0322 108.0217, 123.0452, 231.0663, 272.0326 267.0299, 293.0455, 307.0612, 309.0405 137.0244, 176.0843, 298.0847 109.0295, 497.3272 329.2122, 409.1657 465.3374, 481.3323, 555.3480 108.0217, 109.0295, 465.3374

* CCS for major conformer

30


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-H -H

224.2 309.1

-H -H

222.4 224.5

-H

221.9

-H

224.9

-H -H -H -H -H -H -H -H

220.4 218.5 220.9 219.1 143.2 190.3 169.9 173.6

-H -H -H -H -H

183.2 249.8 249.4 249.2 248.6

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Table 4. CCS determination of the major conformer of six selected compounds in G. buchananii bark extract and solvents used. analysis

compoud intradaya

CCS expected CCS observed 1 2 3 4 5 6 SD [%]

2 198.6 200.5 199.9 199.7 198.5 197.3 196.8 0.8%

21 224.9 226.1 225.3 224.7 223.7 223.1 223.4 0.6%

18 222.4 224.1 223.7 223.3 222.4 222.4 222.6 0.5%

17 224.2 224.9 224.2 224.5 224.0 222.9 223.2 0.4%

20 221.9 222.8 219.5 221.3 221.8 222.2 221.3 0.6%

34 248.7 250.5 250.4 250.2 249.9 248.5 248.8 0.6%

221.9 223.0 221.5 222.6 222.0 222.7 0.9%

220.6 220.4 220.1 221.0 220.4 220.5 0.7%

248.9 247.6 248.2 248.5 248.6 249.9 0.3%

interdayb

CCS observed 1 2 3 4 5 6 SD [%]

198.6 198.8 199.4 199.6 199.1 198.0 0.3%

223.4 223.5 223.7 223.8 223.1 223.8 0.7%

221.9 222.1 222.3 221.3 222.0 222.1 0.3%

concentrationsc

0.1 µl 1.0 µl 10.0 µl SD [%]

CCS observed 1 2 3 4 5 6 7 SD [%] run 1 2

n.d. 198.8 198.7 0.1%

199.0 198.0 197.2 198.4 196.9 196.7 198.4 0.6%

226.1 225.1 223.7 0.5%

224.2 224.5 223.7 223.2 224.0 223.6 224.1 0.5%

223.9 222.6 222.4 0.5%

223.9 223.9 224.2 0.1%

different

solventse

222.6 221.6 220.5 220.0 220.8 220.4 222.1 0.8%

223.7 224.0 223.1 221.9 222.7 222.2 223.3 0.7%

A 0.1% FA 0.1% FA

n.d. 222.1 221.8 0.1%

221.5 222.1 219.8 219.9 220.3 220.3 222.5 0.7% B ACN MeOH

31


n.d. 249.7 248.9 0.4%

250.3 248.5 248.6 247.5 250.3 246.4 249.5 0.6%


3 4 5 6 7

H 2O 1% FA 1% FA H 2O 1% FA

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ACN ACN 1% FA in ACN MeOH 1% FA in MeOH

Standard Deviation, SD [%]; a six injections; b six injections 11 days later; c 0.1, 1 and 10 µl injections; d n.d. not detected; e the following solvents were used.

Table 5. CCS and ratio of CCS of selected compounds. compound ESI neg

CCS peak 1

response peak 1

ratio peak 1 %

CCS peak 2

response peak 2

ratio peak 2 %

21 18 23 4 17 22 20 16 8 19 12 1 5 2

223.7 222.4 218.9 205.1 224.3 221.1 221.8 221.1 212.6 223.6 205.1 204.8 194.3 198.7

2694701 6285892 1459946 1614750 7796276 583662 2495813 905930 122314 2086970 31488 1002479 282444 3499230

94.9 86.1 92.8 97.1 95.3 91.3 95.5 90.2 2.6 98.3 11.1 94.9 80.6 72.3

336.8 330.3 300.3 289.1 309.0 320.7 303.7 319.3 261.7 326.2 258.2 283.0 287.5 283.3

144546 1012608 113727 48675 382972 55604 117994 98850 4633952 35391 251342 54242 68039 1338972

5.1 13.9 7.2 2.9 4.7 8.7 4.5 9.8 97.4 1.7 88.9 5.1 19.4 27.7

ESI pos 17

226.37

5143482

94.7%

310.95

287531

5.3%

32


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FIGURE LEGENDS Figure 1. Chemical structures of compounds 12 and 13. Figure 2. Correlation between mass of G. buchananii compounds and the TWIMS-derived CCS values. Figure 3. Drift time distributions of (A) 17a and 17b and (B) nonamethly-17. Figure 4. Drift time distributions of (A) 13a and 13b, (B) 12a and 12b and (B) 8a and 8b. Figure 5. Drift time distributions of (A) 17a and 17b and (B) 8a and 8b. Figure 6. Drift time distributions of (A) 2a and 2b and (B) 5a and 5b.

33



Figure 1

34


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Figure 2

35



Figure 3

36


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Figure 4

37



Figure 5

38


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Figure 6

39



for TOC only

40


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Construction and Application of a Database for a Five-Dimensional

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