Flavoalkaloids with a Pyrrolidinone Ring from Chinese Ancient

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Bioactive Constituents, Metabolites, and Functions

Flavoalkaloids with Pyrrolidinone Ring from Chinese Ancient Cultivated Tea Xi-Gui Jian Cheng, Fei-Hua Wu, Pu Wang, Jia-Ping Ke, Xiao-Chun Wan, Ming-Hua Qiu, and Guan-Hu Bao J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b02266 • Publication Date (Web): 05 Jul 2018 Downloaded from http://pubs.acs.org on July 7, 2018

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Journal of Agricultural and Food Chemistry

Flavoalkaloids with Pyrrolidinone Ring from Chinese Ancient Cultivated Tea Xi-Gui Jian Cheng1, Fei-Hua Wu3, Pu Wang3, Jia-Ping Ke1, Xiao-Chun Wan1, Ming-Hua Qiu2,*, Guan-Hu Bao1,* 1

Natural Products Laboratory, International Joint Laboratory of Tea Chemistry and

Health effects, State Key Laboratory of Tea Plant Biology and Utilization, Anhui Agricultural University, 230036, Hefei, People’s Republic of China 2

State Key Laboratory of Phytochemistry and Plant Resources in West China,

Kunming Institute of Botany, Chinese Academy of Sciences, Kunming 650201, People's Republic of China. 3

School of Traditional Chinese Pharmacy, China Pharmaceutical University, Nanjing

211198, People's Republic of China.

*

Telephone: +86-0871-5223327.

Fax: +86-0871-5223325,

E-mail:

Fax:

E-mail:

[email protected] *

Telephone:

+86-551-65786401.

+86-551-65786765.

[email protected]. 1

ACS Paragon Plus Environment


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2

Abstract: The Chinese Xi-Gui tea is one ancient cultivated variety of Camellia

3

sinensis var. assamica. It has been used for producing expensive and elite tea in China

4

presently.

5

(−)-6-(5'''S)-N-ethyl-2-pyrrolidinone-epicatechin-3-O-gallate

6

pyrrolidinone

7

(−)-6-(5'''R)-N-ethyl-2-pyrrolidinone-epicatechin-3-O-gallate (etc-pyrrolidinone F，2)

8

(−)-8-(5'''S)-N-ethyl-2-pyrrolidinone-epicatechin-3-O-gallate (etc-pyrrolidinone G, 3a),

9

(−)-8-(5'''S)-N-ethyl-2-pyrrolidinone-catechin-3-O-gallate (etc-pyrrolidinone I, 4a),

10

(−)-8-(5'''R)-N-ethyl-2-pyrrolidinone-catechin-3-O-gallate (etc-pyrrolidinone J, 4b),

11

and

12

(−)-8-(5'''R)-N-ethyl-2-pyrrolidinone-epicatechin-3-O-gallate (etc-pyrrolidinone H，3b)

13

together with the known flavoalkaloids etc-pyrrolidinones A−D (5, 6, 7a，，7b ) were

14

detected and isolated from Xi-Gui green tea. Their structures were identified by

15

comprehensive NMR spectroscopic analyses. Absolute configurations of 1−3 were

16

established by comparison of the CD analyses with epicatechin-3-O-gallate (ECG).

17

Compounds 1−4 were evaluated for their protection against high glucose induced cell

18

senescence on human umbilical vein endothelia cells (HUVECs) and showed

19

significant protection effects (p < 0.01) at both 1.0 and 10 µM. Discussion on the

20

possible evolution of tea plant divergent from related food plants on the basis of

21

phytochemical view was also provided.

22

Keywords: etc-pyrrolidinone, (−)-epicatechin-O-gallate (ECG), Camellia sinensis,

23

(−)-catechin-O-gallate (CG), human umbilical vein endothelia cells (HUVECs)

Five

one

new

E,

new

etc-pyrrolidinone

naturally

occurring

24


flavoalkaloids, (ester-type

catechins

E,

natural

1),

product

Page 3 of 39


25

Introduction

26

Tea, with numerous health benefits as well as special flavor, is one of the most

27

popular non-alcoholic drinks worldwide. The chemical constituents of tea have long

28

been studied, leading to discover a variety of compounds with different structural

29

skeletons.1 Recently, new catechins and their derivatives have been isolated from

30

different types of tea, including the B ring fission catechins from dark tea,

31

phenylpropanoidated catechins from dark tea as well as green tea and its leaves.2 A

32

caffeoylated catechin with impressive anti-acetylcholinesterase activity as well as its

33

neuroprotective effect through interaction with neutrophil gelatinase-associated

34

lipocalin was reported from the special purple tea Zijuan together with its biosynthetic

35

pathway in tea leaves.3,4 Carboxymethyl and carboxyl catechins were also found in

36

Pu-er ripe tea.5 Most interestingly, Wang reported three methylene bridged dimeric

37

imidazole alkaloids from Keemun black tea, which may evidence possible caffeine

38

catabolism pathway in tea plants.6 Additionally, several flavoalkaloids with a

39

pyrrolidinone ring substituted on the A ring of catechins had been found in black,7

40

Pu-er,8 white, and green tea as well as tea leaves.9

41

Flavoalkaloids are a unique class of secondary metabolites with the typical flavonoid

42

skeleton linked with a nitrogen containing 5-membered ring substituted at the position

43

C-6 or/and C-8 of the A ring of flavonoid core structure,10-14 which have attracted

44

academic interest not only in the unique nitrogen containing moieties, but also in the

45

pronounced diversity of biological activity, such as anticancer,15 antiviral properties,16

46

inhibition of advanced glycation end products (AGE),9,17 antioxidant activity,18 etc.



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Flavoalkaloids were found widely distributed in plants. In food materials, they had

48

been found from Actinidia arguta,17 Litchi chinensis,18 besides Camellia sinensis.

49

In tea, a dozen of flavoalkaloids have been reported, all sharing a characteristic ethyl

50

group (-CH2CH3) at the nitrogen atom.7-9 To find more flavoalkaloids from tea, we

51

screened different tea materials by ultra performance liquid chromatography coupled

52

with photodiode array detection and high resolution electrospray ionization mass

53

spectrometry (UPLC-PDA-HR-ESI-MS), indicating that the Chinese ancient

54

cultivated tea Xi-Gui (Xi-Gui) contains relative higher amount of this class of

55

compounds. Xi-Gui (Camellia sinensis var. assamica) grows in Mountain Manglu,

56

Xi-Gui village, Bangdong Town, Lincang County, in Yunnan province.19 Its excellent

57

flavor gets popular with the local residents. However, the low commercial output and

58

special geographical location result in expensive price and no systematic chemical

59

study. Therefore, Xi-Gui green tea was the preferential choice for our continuing

60

study from a natural product approach.

61

UPLC

62

(−)-epicatechin-3-O-gallate (ECG) than that of (−)-epigallocatechin-3-O-gallate

63

(EGCG). Through repeated column chromatography (CC) and comprehensive

64

spectroscopic analyses, we isolated and identified five pairs of flavoallkaloid

65

diastereomers from this Chinese elite tea. In this paper, the isolation, structural

66

elucidation, and protective effects against high dose of glucose induced senescence on

67

human umbilical vein endothelia cells (HUVECs) of the isolated flavoalkaloids (1-4)

68

as well as ECG were presented. The possible reason was also given for the higher

analyses

indicated

that

Xi-Gui

has


a

higher

amount

of

Page 5 of 39


69

content of pyrrolidinonated ECG than that of EGCG in this cultivar. Additionally, we

70

discussed the evolutionary relationship between Actinidia arguta (Chinese kiwifruit)

71

and Camellia sinensis on the basis of the similarity and difference in the structure of

72

flavoalkaloids isolated from these species.

73

Materials and Methods

74

Chemicals. (+)-Catechin (C), (−)-gallocatechin (GC), (−)-catechin-3-O-gallate (CG),

75

and (−)-gallocatechin-3-O-gallate (GCG) standards were purchased from Chengdu

76

Pury Technology Co. Ltd (Chengdu, China). (−)-Epicatechin (EC), EGCG, ECG, and

77

(−)-epigallocatechin (EGC) were purified and identified in our laboratory. Analytical

78

grade of petroleum ether, ethyl acetate, methanol, and dichloromethane were

79

purchased from Chengdu Kelong Chemical Reagent Co., Ltd (Chengdu, China).

80

HPLC grade of methanol, acetonitrile, and formic acid were purchased from Duksan

81

Pure Chemicals Co., Ltd (Ulsan, Korea). HUVECs were bought from Shanghai

82

Honsun Bio Co. (Shanghai, China). Dulbecco’s minimum essential medium (DMEM,

83

Lot No. 1791930) was from Gibco Co. (CA, U.S.A.). Fetal bovine serum (FBS, Lot

84

No. 086005041) was from Genetimes Technology, Inc. (Shanghai, China). Beyotime

85

Institute of Biotechnology (Songjiang, Shanghai) provided the β-galactosidase

86

staining kit for cell senescence. The materials included MCI-Gel CHP20P (Mitsubishi

87

Ltd., Japan), silica gel (Yantai Jiangyou Silicon Development co., Ltd., Shandong,

88

China), Toyopearl HW-40F (Tosoh Bioscience Shanghai Co., Ltd., Shanghai, China),

89

Sephadex LH-20 (GE Healthcare Bio-Sciences AB, Sweden), ODS C-18 (ODS, Fuji

90

Silysia Chemical Ltd., Japan) were filled in open column chromatography (CC) to



91

purify compounds for this study.

92

HPLC semi-preparation was performed on a Waters e2695 combined with a Waters

93

2998 PDA detector (Waters, Milford, Massachusetts, U.S.A.). The semi-preparative

94

column was X Bridge Prep C18 (10 × 250 mm i.d., 5 µm) (Waters, Wexford, Ireland).

95

An FTIR-650 spectrometer was used to observe IR spectrum which purchased from

96

Tianjin Gang Dong Sci. & Tech. Development Co., Ltd (Tianjin, China). Optical

97

rotation was measured on MCP 100 modular circular polarimeter (Anton Paar GmbH,

98

Graz, Austria). 1H and 13C NMR, HMBC, HSQC, ROESY, and 1H-1H COSY spectra

99

were recorded with a DD2 (600 MHz) spectrometer in DMSO-d6 (Agilent Inc., Santa

100

Clara, CA, U.S.A.). CD spectra were measured by a Jasco-810-CD apparatus (Jasco,

101

Tokyo, Japan). Mass spectra were performed on Agilent 1290 UPLC with a

102

photodiode detector array (PDA) coupled to a 6545 time-of-flight (TOF) mass

103

spectrometer with electrospray ionization (ESI) source (Agilent Inc., Santa Clara, CA,

104

U.S.A.). The melting point (m.p.) was observed on SGWX-4 micromelting point

105

apparatus purchased from Beijing Century Science Instruments Co., Ltd (Beijing,

106

China).

107

Tea Materials and Extraction for UPLC Quantification. Xi-Gui was bought from

108

Mount Manglu, Xi-Gui Village, Bangdong, Lincang County, Yunnan province, China,

109

in 2016. The other teas Longjingchangye, Shuchazao, Zijuan, Lu´an GuaPian, Kuding,

110

Fuding-Dabai were plucked in May 2017 from the tea base of Anhui Agricultural

111

University (Hefei, Anhui, China). The detailed extraction method can be found in the

112

literature.20 Briefly, ground tea powder 0.5 g was extracted in 20 mL 70% aqueous


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113

methanol by ultrasonic extraction. The tea extract were centrifuged at 10k rpm and

114

then run through a 0.22 µm filter to store the supernatant.

115

UPLC Quantification of Major Catechins and Caffeine in Xi-Gui

116

The gradient elution of mobile phase A was 0.17% aqueous acetic acid and mobile

117

phase B was acetonitrile in UPLC (Waters, USA) analysis. Details about UPLC

118

method can be found at our previous paper.20 Each sample was repeated trice. The

119

calculated regression equation, relative standard deviation (RSD, including

120

repeatability and reproducibility), correlation coefficient, limit of detection (LOD),

121

limit of quantification (LOQ), and the recovery ratio was also same as those in

122

literature.4

123

Extraction and Isolation. Xi-Gui (5 kg) was ground and extracted by petroleum

124

ether, ethyl acetate, and methanol (each for five times), successively.3 The methanol

125

extract was concentrated under reduced pressure at room temperature to afford a

126

methanol-soluble residue (805 g). This methanol residue was mixed into

127

dichloromethane (1:3, v/v) to provide dichloromethane-soluble fraction and an

128

aqueous phase (593 g). The aqueous phase was concentrated and subjected to

129

MCI-Gel CHP20P CC (water/methanol = 1:0−0:1) to obtain fractions A1−A21 and

130

then the fraction A7 (96 g) was subjected to Toyopearl HW-40F CC and eluted with

131

methanol/water (4:6, v/v), to yield eight fractions (C1 to C8). Fraction C8 (26 g) was

132

then subjected to Sephadex LH-20 CC (methanol/water = 1:9−0:1) to provide

133

fractions D1−D30. D19 was performed on repeated HPLC eluted with gradient

134

acetonitrile/water (gradient elution of acetonitrile was set as follows: 0−35 min, 19%)



135

to produce compound 1 (85 mg), 2 (80 mg), 3 (105 mg), 4 (116 mg), 5 (15 mg), 6 (8

136

mg), 7 (35 mg), respectively (Figure S1).

137

UPLC−ESI-MS/MS Analysis. UPLC−ESI−MS/MS analysis was performed on an

138

Agilent 1290 UPLC instrument with a PDA coupled to a 6545 time-of-flight (TOF)

139

mass spectrometer with ESI source in negative mode. The analysis was obtained by

140

using an ACQUITY UPLC® BEH Shield RP18 column (2.1 × 150 mm, i.d., 1.7 µm)

141

and Agilent qualitative analysis software for data acquisition. The solvent system and

142

the gradient elution condition are same as that in literature.9 The tea sample was

143

gained by ultrasonic extracting 0.25 g of tea powder after ground in 10 mL of

144

methanol for five times within 10 h (20 min each time) for UPLC−ESI−MS/MS

145

analysis. Mass spectra were achieved in full scan MS mode from m/z 100 to 1700.

146

Assay for Protection against High Dose Glucose Induced Senescence on HUVECs.

147

Healthy HUVEC cells were transferred into cell culture bottles under sterile

148

conditions. 10% FBS DMEM medium was placed in a 5% CO2 incubator and

149

incubated at 37 °C. The culture medium was changed once every 1-2 days. After 3-5

150

days of inoculation, the cells grew densely in monolayer and fused and island shaped.

151

When the cells were grown to the fusion stage, they were digested by 0.25% trypsin

152

and passaged by 1:4.21

153

Compounds 1−4, and ECG were dissolved in DMSO, and then diluted to the desired

154

concentrations (1.0 and 10 µM) with the medium, and the final concentration of

155

DMSO was not more than 0.1%. Metformin (300 µM) was used as the positive

156

control. HUVEC cells from logarithmic growth phase were digested into single cell


Page 8 of 39

Page 9 of 39


157

suspension, and 10% FBS DMEM medium was suspended and inoculated in 6-well

158

plates. It was divided into normal group, model group, and drug group. The normal

159

group was HUVECs and the model group was HUVECs co-cultured with 33 mM

160

glucose, and the drug group was HUVECs co-cultured with high glucose plus tested

161

compounds. After 48 h, the culture supernatant was abandoned. Each group was

162

operated according to the instruction of the β-galactosidase staining kit. Finally, the

163

cell senescence was observed by inverted fluorescence microscope (model,

164

IX51S8F-3, from Sigma).

165

Statistical Analysis

166

All assay experiments were obtained ≥ triplicate and the values were presented as

167

mean ± SD. One way ANOVA with Turkey tests was applied to determine significant

168

differences (*P < 0.05, **P < 0.01, ***P < 0.001). GraphPad Prism (version 6.0)

169

software was applied for statistical analysis.

170

Results and Discussion

171

UPLC Quantification of Major Catechins and Caffeine in Xi-Gui tea

172

To quantify the major constituents from Xi-Gui and other green tea, we used UPLC

173

method (Figure 1).20 Table 1 shows that Xi-Gui has the same 8 major catechins as

174

those from other teas with comparative amount. It has the strikingly highest amount of

175

ECG among all the tested samples，which is even significantly higher than that of

176

EGCG (P < 0.01). ECG may help the heart failure patients through improving

177

contractile function as well as reducing pathological remodeling in the dysfunctional

178

heart, suggesting that Xi-Gui tea with the highest amount of ECG may be useful in



179

decreasing cardiac mortality and could be developed as related healthy products.22

180

However, the amount of EGCG, EGC, and EC from Xi-Gui is the smallest among all

181

the samples. Additionally, it also has the highest amount of caffeine, C, which

182

followed by the amounts of GC and CG.

183

Isolation and Identification of Flavoalkaloids from Xi-Gui. Xi-Gui was extracted

184

by petroleum ether, ethyl acetate, and methanol, successively, for phytochemical

185

investigation. The methanol-soluble extract was concentrated and then extracted with

186

dichloromethane. The aqueous residue was fractionated by opened CC and

187

semi-preparative HPLC to provide six flavoalkaloids (Figure 2), two new pure

188

compounds (1 and 2) and two pairs of isomers (3a and 3b, 4a and 4b) in addition to

189

the known flavoalkaloids etc-pyrrolidinones A−D (5, 6, 7a, 7b).9

190

Compounds 1−4 showed the similar IR spectrum, which suggested the presence of

191

hydroxyl groups (broad peak around 3397 cm-1), carbonyl group (around 1695 cm-1),

192

aromatic rings (around 1620, 1520 cm-1).9 Their ESI−HR−MS showed the same

193

deprotonated molecular ion peak at m/z 552.1551 [M-H]− (calcd for 552.1506)

194

corresponding to the molecular formula C28H27NO11, with 16 degrees of unsaturation.

195

The odd number of the molecular weight (553) also suggested the presence of a

196

nitrogen atom in the molecule. The UV λmax (MeOH) peaks of compounds 1−4 are at

197

around 207, 278 nm (Figure S1). The 1H and 13C NMR data of the isomers 1−4 are

198

nearly the same (Table 2 and 3). Above experimental results suggest that these

199

flavoalkaloids are diastereomers.

200

Compound 1 was observed as a white amorphous powder, m.p.: 204-205 °C, [α]25D


Page 10 of 39

Page 11 of 39


201

-65.1 (c 0.1, methanol). The 1H and

13

202

existence of an ECG skeleton in the molecule could be easily deduced from the 1H

203

NMR spectrum compared with that of ECG (Table 2, Figure S2 & S3). The typical

204

proton signals for rings A, B, and C are similar to those of ECG (Table 2), at δH 5.07

205

(1H, br s, H-2), 5.38 (1H, br s, H-3), 3.04 (1H, m, H-4β), 2.79 (1H, d, J = 16.8 Hz,

206

H-4α) (ring C), 6.86 (1H, d, J = 1.2 Hz, H-2'), 6.66 (1H, d, J = 8.4 Hz, H-5'), 6.75 (1H,

207

dd, J = 1.2 Hz, 8.4 Hz, H-6') (ring B), 6.01 (1H, s) (ring A), 6.82 (2H, s) (galloyl-2H),

208

respectively. A single proton signal at A-ring suggested H-6 or 8 of was substituted.

209

Besides signals from ECG unit, the 1H (δH at 2.26 and 2.40 m for H-3''', 2.01 and 2.26

210

m for H-4''', 5.23 m for H-5''', 2.41 and 3.39 m for H-6''', 0.89 for H-7''') and 13C NMR

211

spectra showed signals attributable to a carbonyl (δC 173.3, C-2'''), two methylenes (δC

212

31.1, 23.3, C-3''' and 4'''), a methine (δC 51.5, C-5'''), and an ethyl group (δC 34.3, 12.5,

213

C-6''' and 7'''). The presence of a partial structure of −CH2−CH2−CH− can be deduced

214

by the 1H−1H COSY correlations. HMBC spectrum indicated that the methine carbon

215

(δC 51.5, C-5''') were correlated with the methylene signals (δH 3.39, 2.41, H-6''') at

216

the N-ethyl group. The methylene protons (δH 2.26, 2.40 H-3''') were correlated with

217

the carbonyl carbon (δC 173.3, C-2'''). These HMBC and 1H−1H COSY correlations

218

outlined the presence of an N-ethyl-2-pyrrolidinone ring (Figure 3).9 The only one

219

strong ROESY correlation of the signal (δH 6.01, H-8) with 7-OH proton (δH 9.32)

220

(Figure S11) revealed that the N-ethyl-2-pyrrolidinone moiety was attached at C-6

221

position, which can also be demonstrated by the HMBC correlations between H-5'''

222

(δH 5.23) and C-5 (δC 154.5), C-7 (δC 155.7) (Figure 3). The key HMBC correlations

C NMR data of 1 are shown in Table 2. The



Page 12 of 39

223

between H-4 (δH 2.79) and C-5, 9, and 10 (δC at 154.5, 154.6, 98.4, respectively) can

224

also assign the NMR data of related positions (Figure 3). Thus, the structure of 1 can

225

be decided as 6-N-ethyl-2-pyrrolidinone ECG.

226

Compound 2 was obtained as a white amorphous powder, m.p.: 206-207 °C, [α]25D

227

-135.6 (c 0.1, methanol). Comparing the ESI-HR-MS, 1H, and 13C NMR spectra with

228

those of 1 and ECG (Table 2), compound 2 was determined to be an isomer of 1. The

229

strong ROESY correlation of the signal at A−ring (δH 6.03, H-8) with 7-OH proton

230

(δH 9.45), and the signals (δH 2.76, 3.02, H-4α, 4β) with 5-OH proton (δH 8.55)

231

(Figure S19) both revealed the attachment of N-ethyl-2-pyrrolidinone group at C-6

232

position, indicating that the proton signal (δH 6.03) belongs to H-8 (Table 2).23

233

Besides the similar key HMBC correlations to those of compound 1, correlations

234

between 5-OH (δH 8.55) and C-5, 6, and 10 (δC at 154.9, 107.1, 98.8, respectively)

235

were also observed in the HMBC spectrum of compound 2 (Figure 3).23

236

The absolute configurations at C−2/3 of both flavoalkaloids (1 and 2) were confirmed

237

to be 2R, 3R by comparing NMR data as well as CD curves with that of ECG (Figure

238

S40).8,9,24 Thus, compounds 1 and 2 share the same structure core 6-pyrrolidinone

239

ECG with the only difference at the position C-5'''. The CD spectra of 1 and 2 were

240

compared after subtracting the CD spectrum from each other (Figure 4A).8,9,24 For 1,

241

the arithmetically isolated CD curves of C-5''' showed a strong positive cotton effect

242

(CE) at 211 nm (∆ε＋21.0) and was determined to be of the 5'''S-configuration.

243

Therefore,

244

(−)-6-(5'''S)-N-ethyl-2-pyrrolidinone-epicatechin-3-O-gallate

the

structure

of

compound

1

was


determined and

to

named

be as

Page 13 of 39


245

etc-pyrrolidinone E. Meanwhile, compound 2 presented a negative CE at 211 nm (∆ε

246

− 21.0) comparing with that of 1 by the arithmetically subtracted CD curves (Figure

247

4A) and thus was determined to have a 5''' R-configuration. Therefore, the structure of

248

compound

249

(−)-6-(5'''R)-N-ethyl-2-pyrrolidinone-epicatechin-3-O-gallate

250

etc-pyrrolidinone F.

251

Compounds (5"'S) 3a and (5"'R) 3b are a mixture (Figure S20) in the format of

252

colorless amorphous powder, m.p.: 203-204 °C, [α]25D -168.6 (c 0.07, methanol). The

253

1

254

Furthermore, 3a and 3b can be identified by 2D NMR spectroscopy with similar

255

analyses to those of 1 and 2 (Figure S43). Additionally, the ROESY correlations of

256

both protons (3a: δH 6.04, 3b: 6.09) with 5-OH and 7-OH proton (δH 9.40, δH 9.24)

257

(Figure S27) confirmed that the two protons are both H-6, and thus, the

258

N-ethyl-2-pyrrolidinone group at 3a and 3b were both decided to be linked to the C-8

259

position.

260

(−)-8-N-ethyl-2-pyrrolidinone-epicatechin-3-O-gallate. The positive CE around 203

261

nm (∆ε＋11.1) of compound 3 apparently originated from the chirality of 5'''-S

262

(Figure S40A).24 Since both compound 3 and ECG have the same CE contribution

263

from C-2R and 3R, we also used the same subtracting method to establish the only

264

remaining stereocenter at C-5''' of compound 3 through subtracting the CD spectrum

265

of ECG from that of compound 3. At the same time, the C-5'''R and S can contribute

266

opposite CE,24 the substracting result should be from the remaining stereocenter of the

H and

was

2

determined

to and

be named

as

13

C NMR spectra of 3a and 3b show similarity to those of 1 and 2.

So,

compounds

3a

and

3b

were


determined

to

be


Page 14 of 39

267

major compound.24 Compound 3 (major is 3a) presented a positive CE at 207 nm (∆ε

268

+17.1) comparing with that of ECG8,9,24 (Figure 4B). Thus the major one 3a was

269

determined to have a 5'''S-configuration and named as etc-pyrrolidinone G while the

270

minor

271

(−)-8-(5'''R)-N-ethyl-2-pyrrolidinone-epicatechin-3-O-gallate

272

etc-pyrrolidinone H.

273

Compounds (5"'R) 4a and (5"'S) 4b are a colorless amorphous powder, m.p.:

274

206-207 °C, [α]25D -330.8 (c 0.1, methanol). Compounds 4a and 4b are also a mixture

275

(Figure S28). The negative [α]25D value suggested that compound 4 should have a 3R

276

configuration as that of compounds 1-3 too,25 because a 3S configuration will

277

contribute a positive value, indicating that it possesses an ECG or CG skeleton.

278

However, it cannot possess an ECG skeleton because 1-3 are all the diastereomers

279

with the ECG skeleton already. The key ROESY correlations between H-2 and both

280

H-4α and 4β can establish the α orientation of H-2, different from those of

281

compounds 1−3, and 5−7 which all only have the ROESY correlations between H-2

282

and H-4β, indicating that they all share a β orientation of H-2 (Figure 5, S42),

283

suggesting that compound 4 has a CG skeleton. Additionlally, the HPLC appearance

284

(Figure S1), 1H and 13C NMR spectra of compounds 4a and 4b were similar to those

285

of CG (such as that CG was eluted later then ECG in the HPLC spectrum and

286

8-pyrrolidinone CG was also eluted later than 8-pyrrolidinone ECG, Figure 1).25-28

287

Thus, in combination with other 2D NMR correlations including COSY and HMBC

288

(Figure

one

S43),

3b

compound

was

4

determined

was


and

as named

established

as

as

Page 15 of 39


289

(−)-8-N-ethyl-2-pyrrolidinone-catechin-3-O-gallate, sharing a CG skeleton. Using the

290

same CD spectrum subtracting method as we analyzed with the case of compound 3,

291

compound 4 (major is 4a) presented a negative cotton effect at 212 nm (∆ε -21.3)

292

comparing with that of CG by the arithmetically subtracted CD curves (Figure 4B).

293

The major compound 4a was thus determined to have a 5'''R-configuration and named

294

as etc-pyrrolidinone I. The minor one 4b was determined as 5'''S-configuration and

295

named as etc-pyrrolidinone J.

296

To further study flavoalkaloids from tea, we try to screen different tea materials to

297

find a tea variety with high content of this class of compounds, leading to find that

298

Xi-Gui is the highest one with an amount of about 100 ppm compared to several ppm

299

in white tea Fuding-Dabai on the basis of the isolated quantity.9 However, complete

300

separation of all the diastereomers are not achieved yet since flavoalkaloids tend to

301

appear as pairs of diastereomers naturally.9,17,18,24 It seems those with pyrrolidinone

302

substituted at the position C-8 are always difficult to be separated. Presently, we have

303

only got four completely pure 6-pyrrolidinonated flavoalkaloids. We also found that

304

the contents of these flavoalkaloids may be related to that of the structure core

305

compounds. For example, the highest amount of ECG (Table 1) may contribute to the

306

highest amount of pyrrolidinonated ECG in Xi-Gui, while the relative highest EGCG

307

leads to the correspondingly highest pyrrolidinonated EGCG in Fuding-Dabai.9

308

UPLC−ESI-MS/MS Detection of the Isolated Flavoalkaloids in Xi-Gui. In this

309

study, the isolated six flavoalkaloids (1−4b) can be detected in Xi-Gui by

310

UPLC−ESI-MS/MS analysis guided by the selected mother ion (deprotonated



311

molecular weight m/z 552.1545 [M-H]−) (Figure 6). The result shows that these six

312

flavoalkaloids (1−4b) exist originally in Xi-Gui (Figure 6A), which also implies that

313

the original flavoalkaloids present in tea are not only in the epi-type such as

314

pyrrolidinonated EGCG and ECG,9 but also in other type such as pyrrolidinonated CG,

315

although the amount is different. The fragmental peaks at 400, 382, 236, 169, 125

316

(Figure 6B) in the MS/MS spectrum correspond to the elucidated fragmental

317

structures as shown in Figure 6C, which further confirmed the identified

318

structures.8,9,29

319

Protection against High Dose Glucose Induced Senescence on HUVECs by

320

Flavoalkaloids 1−4.

321

The β-galactosidase was located in cytoplasm, and the positive product was blue. The

322

rate of β-galactosidase positive cells in the cell aging model group (high glucose

323

group, HG, 30 mM) was significantly higher than that in the normal control group (P

324

= 0.000). Figure 7 implies that the positive control methformin (300 µM) and all

325

pyrrolidinonated ECG (1−4) together with ECG (1.0 and 10µM) can prevent

326

endothelia dysfunction and be helpful in protection against vascular cell aging (P

Flavoalkaloids with a Pyrrolidinone Ring from Chinese Ancient

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