Novel Theaflavin-Type Chlorogenic Acid Derivatives Identified in

Subscriber access provided by - Access paid by the | UCSB Libraries

Bioactive Constituents, Metabolites, and Functions

Novel Theaflavin-type Chlorogenic Acid Derivatives Identified in Black Tea Shuwei Zhang, Chun Yang, Emmanuel Idehen, Shi Lei, Lishuang Lv, and Shengmin Sang J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b06044 • Publication Date (Web): 13 Mar 2018 Downloaded from http://pubs.acs.org on March 17, 2018

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 24

Journal of Agricultural and Food Chemistry

1

Novel Theaflavin-type Chlorogenic Acid Derivatives Identified in Black Tea

2

Shuwei Zhang†, Chun Yang †, ‡, Emmaneul Idehen†, Lei Shi†, ‡, Lishuang Lv§, and Shengmin

3

Sang†, *

4 †

5

Laboratory for Functional Foods and Human Health, Center for Excellence in Post-Harvest

6

Technologies, North Carolina Agricultural and Technical State University, North Carolina

7

Research Campus, 500 Laureate Way, Kannapolis, NC 28081, USA

8

‡

Department of Colorectal Surgery, General Hospital of Ningxia Medical University, Yinchuan 750004, P.R. China

9 10

§

Department of Food Science and Technology, Nanjing Normal University, 122# Ninghai Road, Nanjing, 210097, P. R. China

11 12 13

*Corresponding Author (Tel: 704-250-5710; Fax: 704-250-5729; E-mail: [email protected]

14

or [email protected])

15

ACS Paragon Plus Environment

1


Page 2 of 24

16

ABSTRACT

17

Consumption of black tea contributed to many health benefits including the prevention of heart

18

disease and certain types of cancer. However, the chemical composition of black tea has not been

19

fully explored. Most studies have examined different interactions between the four major tea

20

catechins, and limited studies have investigated the interaction between catechins and other

21

components in tea. In the present study, we tested our hypothesis that the ortho-dihydroxyl

22

structure of chlorogenic acid (CGA) could react with the vic-trihydroxy structure of (−)-

23

epigallocatechin 3-gallate (EGCG) and (−)-epigallocatechin (EGC) to generate theaflavin-type of

24

compounds during black tea fermentation. The reaction between CGA and EGCG or EGC was

25

catalyzed by horseradish peroxidase (POD) in the presence of H2O2. Two theaflavin-type

26

compounds EGCG-CGA and EGC-CGA were purified using Sephadex LH-20 column. Their

27

structures were elucidated based on the analysis of their MS and 1D- and 2D-NMR spectroscopic

28

data. Furthermore, the existence of these two novel compounds was characterized by LC/MS/MS

29

analysis. We also found that EGCG-CGA and EGC-CGA had very similar inhibitory effects on

30

the growth of human colon cancer cells with that of theaflavin 3,3'-digallate. These findings shed

31

light on the interactions between the major bioactive compounds, catechins, and other minor

32

compounds in tea. The confirmation of the presence of this type of reaction in black tea may

33

provide more understanding of the complexity of black tea chemistry.

34

Keywords: Chlorogenic acid, EGCG, EGC, benzotropolone, enzymatic model reaction

35


2

Page 3 of 24


36

INTRODUCTION

37

Epidemiological studies have associated the regular consumption of tea (Camellia sinensis) with

38

the potential to reduce the risk of many chronic diseases.1-3 These health benefits are attributed to

39

the presence of high amounts of bioactive polyphenols in tea. An impressive number of scientific

40

publications have focused on the four major catechins: (−)-epicatechin 3-gallate (ECG), (−)-

41

epigallocatechin 3-gallate (EGCG), (−)-epicatechin (EC), and (−)-epigallocatechin (EGC).

42

However, besides catechins, other compounds in tea like theaflavins may also possess

43

remarkable bioactive potential.

44

Theaflavins are major constituents of black tea, the most consumed tea among the five

45

major types of teas (black, green, white, oolong, and pu-erh). These compounds have recently

46

gained significant attention due to their various pharmacological, biological, and health

47

promoting benefits. Studies have shown that these compounds may offer positive effects on

48

diseases such as obesity,4, 5 cardiovascular diseases,6-8 Alzheimer,9 cancer,10-12 and may even

49

lower cholesterol levels.13 It is also known that theaflavins considerably contribute to the

50

properties of black tea's briskness, brightness, strength, mouthfeel, color, and the extent of cream

51

formation.14-18

52

The benzotropolone skeleton of theaflavins is formed from co-oxidation of selected pairs

53

of compounds, one with an ortho-dihydroxyl structure and the other with a vic-trihydroxy

54

structure, during fermentation by two enzymes in green tea, peroxidase (POD) and polyphenol

55

oxidase/tyrosinase (PPO).19-22 POD and PPO are key enzymes to produce the reddish color

56

compounds in black tea.23, 24 And many studies have been carried out to confirm the oxidation

57

products in black tea.25, 26 Thus far, most studies have examined different interactions between

58

the four major tea catechins, which could lead to the formation of the major theaflavins: from EC


3


Page 4 of 24

59

and EGC to theaflavin, from EC and EGCG to theaflavin 3-gallate, from ECG and EGC to

60

theaflavin 3'-gallate , and from ECG and EGCG to theaflavin 3,3'-digallate, from ECG and ECG

61

to theaflavate A, and from EC and ECG to theaflavate B .27 Additionally, EGCG and EGC could

62

react with gallic acid to generate theaflavic acid-3'-gallate and theaflavic acid, respectively.27

63

Few studies have examined the interactions between catechins and other components in tea.

64

Chlorogenic acid (CGA), also known as 5-O-caffeoylquinic acid (5-CQA), is the

65

ester formed between caffeic acid and the 5-hydroxyl position of L-quinic acid.28 It is one of the

66

most available acids among phenolic acid compounds, which can be naturally found in green

67

coffee extracts and tea.27, 28 The content of chlorogenic acid in green tea is in the range of 0.01-

68

0.40 mg/g.29 CGA has the ortho-dihydroxyl structure, which has the potential to react with the

69

vic-trihydroxy structure of EGCG and EGC to generate theaflavin-type of compounds during

70

black tea fermentation. However, there is no study on the interaction between CGA and tea

71

catechins in the literature. In the present study, we reported the synthesis of two theaflavin-type

72

compounds EGCG-CGA and EGC-CGA using POD/H2O2 model oxidation system, the

73

elucidation of their structure using NMR, the identification of these two compounds in black tea

74

using LC/MS, and their cytotoxic activities against human colon cancer cells.

75

MATERIALS AND METHODS

76

Materials: Horseradish peroxidase, hydrogen peroxide, sodium citrate and dibasic sodium

77

phosphate were purchased from Sigma (St. Louis, MO). Sephadex LH-20 was purchased from

78

Thermo Fisher Scientific (Waltham, MA). Chlorogenic acid, EGCG and EGC were purchased

79

from Synnavator Inc (Durham, NC). Theaflavin 3,3'-digallate was prepared in our lab.27 LC/MS-

80

grade methanol, water, and formic acid, and ACS grade ethyl acetate and acetone were

81

purchased from VWR Scientific (South Plainfield, NJ). Human colon cancer cells, HCT-116


4

Page 5 of 24


82

were purchased from American Type Tissue Culture (Manassas, VA). MTT (3-(4,5-

83

dimethylthiaxol-2-yl)-2,5-diphenyltetrazolium bromide) was obtained from Calbiochem-

84

Novabiochem (San Diego, CA). Fetal bovine serum (FBS) and penicillin/streptomycin were

85

obtained from Gemini-Bio-Products (West Sacramento, CA). Lipton black tea bags were

86

purchased from local supermarkets.

87

Synthesis of theaflavin-type compounds

88

EGCG-CGA, 4 (Figure 1): The pH 5.0 phosphate-citrate buffer was prepared by mixing

89

0.467 g of citric acid and 0.915 g of dibasic sodium phosphate in 100 mL of water. Then 1 mL of

90

acetone was added into 10 mL of this buffer and mixed completely. EGCG, 1 (250mg) and

91

CGA, 3 (200mg) were dissolved in this mixture, and then added 1 mg of horseradish peroxidase.

92

A 2 mL aliquot of 3.13% H2O2 was added into the mixture drop by drop during 45 min while

93

stirring at room temperature. Ethyl acetate (3 x 50 mL) was used to extract the compounds out.

94

The ethyl acetate extract was dried by rotary evaporator, and the residue was dissolved in ethanol

95

and applied to a Sephadex LH-20 column, which was eluted with acetone water, 2:3 (v/v). The

96

separation was monitored by thin-layer chromatography (TLC) and the fractions with a single

97

red spot were combined and dried to obtain 11 mg of a reddish amorphous powder: 1H- and 13C-

98

NMR (600 MHz, CD3OD) (Table 1); negative ESIMS, m/z 779.3 [M - H]-.

99

EGC-CGA, 5 (Figure 1): Following the procedure for the synthesis of 4, EGC, 1 and

100

CGA, 3 were used to synthesize 13 mg of another reddish amorphous powder: 1H- and 13C-NMR

101

(600 MHz, CD3OD) (Table 1); negative ESIMS, m/z 627.2 [M - H]-.

102

Preparation of black tea extract


5


Page 6 of 24

103

Two bags of Lipton black tea (3.8 g) were extracted with boiling water (100 mL) for 10

104

min. The extract was centrifuged at 16100 ×g for 15 min; the supernatant was transferred into

105

vials for LC/MS analysis.

106

LC/MS Analysis

107

LC-MS analysis was performed with a Thermo-Finnigan Spectra system This system

108

consisted of an Ultimate 3000 RS pump, an Ultimate 3000 degasser, an Ultimate 3000 RS

109

column compartment, an Ultimate 3000 RS autosampler, and an LTQ Velos Pro ion trap mass

110

spectrometer (Thermo Fisher Scientific, Waltham, MA) with an electrospray ionization (ESI)

111

interface. The column used was a 150 mm × 3.0 mm i.d., 5 µm, Gemini C18 (Phenomenex,

112

Torrance, CA) to analyze the synthesized compounds and tea extracts with an injection volume

113

of 10 µL and a flow rate of 0.3 mL/min. Column elution started with 100% solvent A (5%

114

aqueous methanol with 0.1% formic acid), followed by a linear increase to 75% solvent B (95%

115

aqueous methanol with 0.1% formic acid) from 0 to 25 min, 100% B from 25 to 30 min, washed

116

with 100% B from 30-35 min, and then equilibrated to 100% A from 35 to 45 min for the next

117

run. The column temperature was maintained at 35 °C.

118

Each standard was used to tune the mass detector under a negative ESI ion mode. The

119

voltage on the ESI interface was kept at approximately -3.6 kV. Nitrogen gas was used as the

120

auxiliary gas (10 arbitrary units) and the sheath gas (34 arbitrary units). The voltage and capillary

121

temperature was maintained at -45 V and 300 °C. The tube lens offset voltage (120 V) was tuned

122

using authentic theaflavin. MS-MSn (n=2-3) analysis was conducted under selected ion

123

monitoring (SIM) mode.

124

normalized collision energy of 35 values and an isolation width of 1.2 Da. The mass data of all

Collision induced dissociation (CID) was conducted using a


6

Page 7 of 24


125

samples were acquired in the range of m/z 50-1000. Xcalibur 2.0 (Thermo Electron; San Jose,

126

CA) was used for all the mass data.

127

The concentrations of compounds 4, 5, and 6 in black tea extract were measured using

128

SIM mode with the following transitions: 4, m/z 779.3→627.2; 5, m/z 627.2→435.1; and 6, m/z

129

867.3→715.2. The standard solutions of compounds 4, 5, and 6 were made from 0.5µg/mL to

130

10.0µg/mL, 0.25µg/mL to 5.0µg/mL, and 1.0µg/mL to 40.0µg/mL in methanol, respectively. All

131

the samples were analyzed in triplicate. The calibration curves of the three standards exhibited

132

linearity with correlation greater than 0.995.

133

Nuclear Magnetic Resonance (NMR)

134

An AVANCE 600 MHz spectrometer (Bruker Inc., Silberstreifen, Rheinstetten,

135

Germany) was used to record all the NMR data. CD3OD was used as the solvent for all the

136

compounds. The carbon NMR spectra are proton decoupled.

137

MTT Assay

138

Cell growth inhibition was measured by the MTT colorimetric assay.30 HCT-116 human

139

colon cancer cells were plated in 96-well microtiter plates with 8000 cells/well, and cultured in

140

McCoy’s 5A medium containing 1% glutamine, 1% penicillin/streptomycin, and 10% fetal

141

bovine serum, for 24 h in a incubator with 95% humidity and 5% CO2 at 37 °C. The test

142

compounds in DMSO was added to the cell culture medium to make up a final DMSO

143

concentration of 0.1% for both control and treatment groups. After culturing the cells for 24 h,

144

the medium was removed and the cells were treated with 200 µL fresh medium containing 2.41

145

mmol/L MTT. After incubation for 3 h at 37 °C, the medium containing MTT was removed and

146

the formazan precipitate was solubilized by adding 100 µL of DMSO. After gently shaken for

147

one hour at room temperature, the plates were placed into a microtiter plate reader (Biotek,


7


Page 8 of 24

148

Winooski, VT). Absorbance values were recorded at 550 nm and expressed as a percentage of

149

viable cells in the control.

150

RESULTS AND DISCUSSION

151

Synthesis and Structural Elucidation of Compound 4

152

To test our hypothesis that the ortho-dihydroxyl structure of CGA has the potential to

153

react with the vic-trihydroxy structure of EGCG to form the benzotropolone structure of a new

154

theaflavin-type compound, we reacted CGA and EGCG catalyzed by horseradish POD in the

155

presence of H2O2. As expected, a new reddish colored compound (4) was generated, which was

156

purified through a Sephadex LH-20 column. Compound 4 (Figure 1) had the molecular formula

157

C37H32O19 inferred from the negative ESI-MS at m/z 779.3 [M – H]- and NMR data (Table 1). Its

158

NMR spectra suggested signals for a ketone group (δC 184.7 ppm), three oxidized phenolic

159

carbons (δC 157.1, 147.9, and 155.2 ppm), and three phenolic methine carbons (δC 118.0, 126.6,

160

and 121.4 ppm) whose protons showed singlet peaks at δH 8.06 (1H, s), 7.53 (1H, s), and 7.33

161

ppm (1H, s), suggesting the presence of a benzotropolone structure.31 The proton NMR spectrum

162

of 4 exhibited signals at the 2- (δH 5.10 (1H, s)), 3- (δH 5.77 (1H, d, 4.1)), 4- (δH 3.13 (1H, dd,

163

4.8,17.4) and 2.90 (1H, d, 17.0)), 6- (δH 6.02 (1H, d, 2.0)), and 8- (δH 6.19 (1H, d, 1.8)) positions

164

of the flavan-3-ol unit, and its

165

substituted with three oxygen atoms (δC 100.3, 157.5, 96.6, 159.4, 94.8, 158.7 ppm), a C-ring

166

containing two oxidized methines (δC 79.1, 67.5 ppm), and one methene (δC 25.5 ppm) group,

167

indicating that the A and C rings of EGCG remained the same during the oxidation.

168

Additionally, the carbon NMR spectrum of 4 displayed signals for a galloyl unit with six

169

phenolic carbons (δC 122.1, 109.0, 147.0, 140.5, 147.0, and 109.0 ppm) and a carbonyl group (δC

170

168.2 ppm) (Table 1),32 suggesting that there was no change of the galloyl ester group of EGCG.

13

C-NMR spectrum exhibited a meta-substituted A-ring


8

Page 9 of 24


171

Furthermore, the NMR data also showed the presence of the double bond, ester bond, and quinic

172

acid of the CGA with one trans-substituted double bond (δC 142.5 and 120.6 ppm; and δH 8.27

173

(1H, d, 15.5) and 6.18 ppm (1H, d, 15.6)), two carbonyl groups (δC 169.7 and 168.2 ppm), three

174

oxidized methines (δC 71.8, 74.3, and 72.0 ppm), one oxidized quaternary carbon (δC 76.6 ppm),

175

and two methylenes (δC 39.6 and 37.8 ppm). All of the above spectroscopic features suggest that

176

the benzotropolone structure was formed by the B ring of EGCG and the phenyl ring of CGA.

177

This assertion was further supported by HMBC spectrum, which displayed correlation peaks

178

from H-c at δH 7.33 (1H, s) to C-2 (δC 79.1 ppm), C-a (δC 184.7 ppm), C-b (δC 157.1 ppm), and

179

C-d (δC 135.3 ppm), from H-e at δH 8.06 (1H, s) to C-2 (δC 79.1 ppm), C-j (δC 122.9 ppm), C-k

180

(δC 123.6 ppm), and C-f (δC 129.5 ppm), and from H-g at δH 7.53 (1H, s) to C-h (δC 147.9 ppm),

181

C-k (δC 123.6 ppm), and C-i (δC 155.2 ppm) (Figure 2). Thus, the structure of 4 was tentatively

182

deduced as shown in Figure 1. The assignment of 1H and

183

interpretation of the results of HMQC and HMBC experiments (Table 1). This is the first report

184

to synthesize and elucidate the structure of the theaflavin-type EGCG and CGA product.

185

Structural Elucidation of Compound 5

13

C signals of 4 was based on the

186

Similarly, we purified a new reddish colored compound (5) from the reaction between

187

EGC and CGA in the presence of horseradish POD and H2O2. Compound 5 (Figure 1) had the

188

molecular formula C30H28O15 inferred from the negative ESI-MS at m/z 627.2 [M – H]- and

189

NMR data (Table 1), which was 152 mass units (one galloyl unit) less than that of 4. Its 1H- and

190

13

191

signals for one galloyl unit, and the NMR data also indicated the presence of a benzotropolone

192

structure with a ketone group (δC 185.6 ppm), three oxidized phenolic carbons (δC 156.0, 147.0,

193

and 154.1 ppm), and three phenolic methine carbons (δC 119.0, 129.1, and 122.1 ppm) whose

C-NMR spectra were very similar to those of compound 5 except for the disappearance of the


9


Page 10 of 24

194

protons showed singlet peaks at δH 8.05 (1H, s), 7.64 (1H, s), and 7.40 ppm (1H, s). Therefore,

195

the structure of 5 was tentatively deduced as shown in Figure 1.

196

Identification of 4 and 5 in black tea.

197

To investigate whether these enzymatic model reactions could occur in the fermentation

198

process, we searched for the presence of compounds 4 and 5 in black tea extract using LC/MS.

199

Under SIM mode of searching m/z 779.3 [M – H]-, a peak at retention time 28.2 min in black tea

200

extract was observed (Figure 3A). This peak had the same chromatographic retention time,

201

molecular mass, and fragment ion spectrum with those of the authentic standard 4 (Figure 3A).

202

Both of them had m/z 627 as the base peak, which was a typical loss of a galloyl unit (m/z 152).

203

While the fragment ion at m/z 605 was due to the loss of a quinic acid (m/z 192) and one water

204

molecule, which would sequentially lose one CO2 molecule to generate fragment ion at m/z 561

205

(Figure 3A). In addition, the tandem mass of the fragment ion m/z 627 (MS3: 627/779 [M –H]-)

206

of the peak in black tea extract was almost identical to that of the authentic standard 4 (Figure

207

3A). Both of them had m/z 435 as base ion, which lost one quinic acid molecule.

208

Under the search of 5 (m/z 627 [M – H]-), two peaks (26.3 and 27.5 min) appeared in

209

black tea extract. The peak at retention time 27.5 min and the synthetic standard 5 had almost

210

identical retention time and tandem mass spectrum (Figure 3B). They had the fragment ion m/z

211

435 as base ion, which lost one quinic acid molecule. In addition, the MS2 spectrum of 5 (MS2:

212

627 [M – H]-) was the same as the MS3 spectrum of the fragment ion m/z 627 (MS3: 627/779 [M

213

– H]-) of 4 (Figure 3A and 3B), confirming that these two compounds share the same core

214

structure. Interestingly, the peak at retention time 26.3 min also had similar tandem mass

215

spectrum with those of the peak at retention time 27.5 min and the synthetic standard 5 (Figure

216

3B), indicating it was a potential stereoisomer of 5, which needs to be proven experimentally.


10

Page 11 of 24


217

Using the synthesized compounds as standards, the concentrations of 4 and 5 were quantitated

218

and compared with that of 6, one of the major theaflavins in black tea. The concentrations of

219

compounds 4 and 5 in black tea were found to be 41.6 µg/g and 15.3 µg/g, which were much

220

lower than the concentration of 6, 340 µg/g. Altogether, our results clearly indicate that CGA

221

could react with EGCG and EGC during black tea fermentation process to generate two minor

222

theaflavin-type compounds 4 and 5.

223

The growth inhibitory effects of 4 and 5 on human colon cancer cells.

224

Theaflavins are the major polyphenols in black tea and have been confirmed to show

225

different beneficial health effects.33-35 To determine whether two novel theaflavin-type

226

compounds 4 and 5 are biologically active, we studied their inhibitory effects on the growth of

227

HCT-116 human colon cancer cells using 6, one of the major theaflavins in black tea, as the

228

positive control. As shown in Figure 4, both 4 (IC50: 120.5 µM) and 5 (IC50: 110.4 µM) had very

229

similar activities with that of 6 (IC50: 111.4 µM), and could inhibit the growth of HCT-116 cells

230

dose dependently. The similarity in inhibitory activity of 4 and 5 with 6 suggests that the

231

benzotropolone skeleton of theaflavins may play a major role in the anti-cancer activities of

232

theaflavins.

233

In conclusion, the present study confirmed our hypothesis that the ortho-dihydroxy group

234

of CGA could react with the vic-trihydroxyl B ring of EGCG and EGC to form theaflavin-type

235

products with a core benzotropolone skeleton. This is the first study to use enzymatic model

236

reactions to synthesize 4 and 5, to use 1D- and 2D-NMR to elucidate their structures, to use

237

LC/MS to confirm the presence of these two compounds in black tea, and to evaluate their

238

growth inhibitory effects on human colon cancer cells. These findings shed light on the

239

interactions between the major bioactive compounds, catechins, and other minor compounds in


11


Page 12 of 24

240

tea. Besides caffeoylquinic acids, there are several galloylquinic acid derivatives reported in

241

green tea leaf.36 It is worthwhile to further study whether these galloylquinic acid derivatives

242

could reactive with catechins to generate theaflavin-type of compounds. Products from this kind

243

of interaction may further interact with other components of tea to form more complex

244

thearubigins. The confirmation of this type of reaction in black tea also provides more

245

understanding of the complexity of the black tea chemistry.

246

ACKNOWLEDGEMENT

247

The authors wish to thank Mr. Hunter Snooks who assisted in the proofreading of the manuscript.

248

This work was supported by NIH R01 grant AT008623 to S. Sang.

249


12

Page 13 of 24


Reference 250

1.

da Silva Pinto, M., Tea: A new perspective on health benefits. Food Res. Int. 2013, 53,

251

558-567.

252

2.

253

6141-6147.

254

3.

255

Vervoort, J.; van der Hooft, J. J.; Roger, L.; Draijer, R.; Jacobs, D. M., Interactions of black tea

256

polyphenols with human gut microbiota: implications for gut and cardiovascular health. Am. J.

257

Clin. Nutr. 2013, 98, 1631S-1641S.

258

4.

259

lowering effects of theaflavins on high-fat diet induced obese rats. J. Funct. Foods 2013, 5,

260

1142-1150.

261

5.

262

a black tea polyphenol, stimulates lipolysis associated with the induction of mitochondrial

263

uncoupling proteins and AMPK–FoxO3A–MnSOD pathway in 3T3-L1 adipocytes. J. Funct.

264

Foods 2015, 17, 271-282.

265

6.

266

effects of Turkish Black Tea on TA98 and TA100 strains of Salmonella typhimurium (in vitro)

267

and mice (in vivo). Pharm. Biol. 2017, 55, 1202-1206.

268

7.

269

Mochizuki, K.; Nakayama, T.; Osakabe, N., The impact of theaflavins on systemic-and

270

microcirculation alterations: The murine and randomized feasibility trials. J. Nutr. Biochem.

271

2016, 32, 107-114.

Khan, N.; Mukhtar, H., Tea and health: studies in humans. Curr. Pharm. Des. 2013, 19,

van Duynhoven, J.; Vaughan, E. E.; van Dorsten, F.; Gomez-Roldan, V.; de Vos, R.;

Jin, D.; Xu, Y.; Mei, X.; Meng, Q.; Gao, Y.; Li, B.; Tu, Y., Antiobesity and lipid

Ko, H.-J.; Lo, C.-Y.; Wang, B.-J.; Chiou, R. Y.-Y.; Lin, S.-M., Theaflavin-3, 3′-digallate,

Charehsaz, M.; Sipahi, H.; Giri, A. K.; Aydin, A., Antimutagenic and anticlastogenic

Saito, A.; Nakazato, R.; Suhara, Y.; Shibata, M.; Fukui, T.; Ishii, T.; Asanuma, T.;


13


Page 14 of 24

272

8.

Hartley, L.; Flowers, N.; Holmes, J.; Clarke, A.; Stranges, S.; Hooper, L.; Rees, K., PP10

273

green and black tea for the primary prevention of cardiovascular disease (CVD): a cochrane

274

systematic review. J. Epidemiol. Community Health 2013, 67, A52-A53.

275

9.

276

neurocognitive performance of patients with early onset of Alzheimer by Montreal Cognitive

277

Assessment. Int. J. Pharm. Biol. Arch. 2017, 7, 11-16.

278

10.

279

M. N.; Adhikari, A.; Chatterjee, A., Effect of black tea polyphenol on cell-ECM interaction and

280

MMP. Am. J. Plant Sci. 2017, 8, 856-866.

281

11.

282

of black tea pigments, theaflavin‑3/3'-gallate against cisplatin-resistant ovarian cancer cells by

283

inducing apoptosis and G1 cell cycle arrest. Int. J. Oncol. 2017, 51, 1508-1520.

284

12.

285

as anticarcinogens: mechanisms, anti-multidrug resistance and QSAR Studies. Curr. Med. Chem.

286

2012, 19, 4324-4347.

287

13.

288

solubility of cholesterol in vitro and intestinal absorption of cholesterol in rats. J. Agric. Food

289

Chem. 2010, 58, 8591-8595.

290

14.

291

composition of Indian black teas. J. Food Sci. Technol. 2017, 54, 1266-1272.

292

15.

293

in black tea liquors in assessments of quality in teas. Analyst 1961, 86, 94-98.

Mitra, A., Study on effectivity of black tea as adjuvant therapy in improving

Bhattacharyya, N.; Mondal, S.; Moulik, S.; Paul, S.; Bhattacharrya, S.; Hazra, A. K.; Ali,

Pan, H.; Wang, F.; Rankin, G. O.; Rojanasakul, Y.; Tu, Y.; Chen, Y. C., Inhibitory effect

G Mercader, A.; B Pomilio, A., (Iso) flav (an) ones, chalcones, catechins, and theaflavins

Ikeda, I.; Yamahira, T.; Kato, M.; Ishikawa, A., Black-tea polyphenols decrease micellar

Khanum, H.; Faiza, S.; Sulochanamma, G.; Borse, B., Quality, antioxidant activity and

Roberts, E.; Smith, R., Spectrophotometric measurements of theaflavins and thearubigins


14

Page 15 of 24


294

16.

Millin, D. J.; Crispin, D. J.; Swaine, D., Nonvolatile components of black tea and their

295

contribution to the character of the beverage. J. Agric. Food Chem. 1969, 17, 717-722.

296

17.

297

cream formation: the contribution of black tea phenolic pigments determined by HPLC. J. Sci.

298

Food Agric. 1993, 63, 77-86.

299

18.

300

astringent taste of black tea brews. Z. Lebensm. Unters. Forsch. 1992, 195, 108-111.

301

19.

302

Engelhardt, U. H., Investigation of processes in black tea manufacture through model

303

fermentation (oxidation) experiments. J. Agric. Food Chem. 2014, 62, 7854-7861.

304

20.

305

stability. Food Funct. 2013, 4, 10-18.

306

21.

307

human relevance. Annu. Rev. Nutr. 2013, 33, 161-181.

308

22.

309

patterns in relation to theaflavin and thearubigins formation. Food Chem. 2009, 115, 8-14.

310

23.

311

suspension. Influence of tea peroxidase. J. Sci. Food Agric. 1981, 32, 920-932.

312

24.

313

hydroxylation step in the “oxidative cascade” during oxidation of tea catechins. J. Agric. Food

314

Chem. 2016, 64, 8002-8009.

315

25.

316

based on selected ions detects low-abundance phenolics in black tea – theatridimensins as

Powell, C.; Clifford, M. N.; Opie, S. C.; Ford, M. A.; Robertson, A.; Gibson, C. L., Tea

Ding, Z.; Kuhr, S.; Engelhardt, U. H., Influence of catechins and theaflavins on the

Stodt, U. W.; Blauth, N.; Niemann, S.; Stark, J.; Pawar, V.; Jayaraman, S.; Koek, J.;

Li, S.; Lo, C.-Y.; Pan, M.-H.; Lai, C.-S.; Ho, C.-T., Black tea: chemical analysis and

Yang, C. S.; Hong, J., Prevention of chronic diseases by tea: possible mechanisms and

Ngure, F. M.; Wanyoko, J. K.; Mahungu, S. M.; Shitandi, A. A., Catechins depletion

Dix, M. A.; Fairley, C. J.; Millin, D. J.; Swaine, D., Fermentation of tea in aqueous

Verloop, A. J. W.; Vincken, J.-P.; Gruppen, H., Peroxidase can perform the

Verloop, A. J. W.; Vincken, J.-P.; Gruppen, H., A tandem mass spectrometry method


15


Page 16 of 24

317

products of the oxidative cascade. Rapid Communications in Mass Spectrometry 2016, 30, 1797-

318

1805.

319

26.

320

polyhydroxy-theaflavins and theacitrins in the formation of black tea thearubigins: evidence by

321

tandem LC-MS. Food Funct 2010, 1, 180-99.

322

27.

323

tea constituents. Pharmacol. Res. 2011, 64, 87-99.

324

28.

325

F.; Babazadeh, D.; FangFang, X.; Modarresi-Ghazani, F.; WenHua, L.; XiaoHui, Z.,

326

Chlorogenic acid (CGA): A pharmacological review and call for further research. Biomed.

327

Pharmacother. 2017, 97, 67-74.

328

29.

329

Xu, Xiao-ping, Comparison of chlorogenic acid content in different kinds of tea. Huaxi Yaoxue

330

Zazhi 2008, 23, 190-192.

331

30.

332

proliferation and cytotoxicity assays. J. Immunol. Methods 1983, 65, 55-63.

333

31.

334

characterized from black tea using LC/MS/MS. Bioorg. Med. Chem. 2004, 12, 3009-3017.

335

32.

336

of Myrica rubra Var. acuminata. J. Agric. Food Chem. 2003, 51, 2974-2979.

337

33.

338

responses during carcinogenesis. Mini Rev. Med. Chem. 2010, 10, 492-505.

Kuhnert, N.; Clifford, M. N.; Muller, A., Oxidative cascade reactions yielding

Sang, S.; Lambert, J. D.; Ho, C. T.; Yang, C. S., The chemistry and biotransformation of

Naveed, M.; Hejazi, V.; Abbas, M.; Kamboh, A. A.; Khan, G. J.; Shumzaid, M.; Ahmad,

Zhou, S. W., Xiao-dong; Liu, Jing; Zhang, Jie; He, Ying-jin; Tian, Xin; He, Wen-wen;

Mosmann, T., Rapid colorimetric assay for cellular growth and survival: application to

Sang, S.; Tian, S.; Stark, R. E.; Yang, C. S.; Ho, C. T., New dibenzotropolone derivatives

Yang, L.-L.; Chang, C.-C.; Chen, L.-G.; Wang, C.-C., Antitumor Principle Constituents

Kumar, G.; Pillare, S. P.; Maru, G. B., Black tea polyphenols-mediated in vivo cellular


16

Page 17 of 24


339

34.

Sharma, V.; Rao, L. J., A thought on the biological activities of black tea. Crit. Rev. Food

340

Sci. Nutr. 2009, 49, 379-404.

341

35.

342

of black tea pigments, theaflavin3/3'-gallate against cisplatin-resistant ovarian cancer cells by

343

inducing apoptosis and G1 cell cycle arrest. Int. J. Oncol. 2017, 51, 1508-1520.

344

36.

345

molecular weight black tea polyphenols. Nat. Prod. Rep. 2010, 27, 417-62.

Pan, H.; Wang, F.; Rankin, G. O.; Rojanasakul, Y.; Tu, Y.; Chen, Y. C., Inhibitory effect

Drynan, J. W.; Clifford, M. N.; Obuchowicz, J.; Kuhnert, N., The chemistry of low

346


17


Page 18 of 24

FIGURE LEGENDS Figure 1. Structures of epigallocatechin 3-gallate (EGCG, 1), epigallocatechin (EGC, 2), chlorogenic acid (CGA, 3), and the benzotropolone products EGCG-CGA (4), EGC-CGA (5), and theaflavin 3,3'-digallate (6). Figure 2. Key HMBC and 1H-1H COSY correlations of EGCG-CGA, 4. Figure 3. LC chromatograms and tandem mass spectra of (A) EGCG-CGA, 4 and (B) EGCCGA, 5. Figure 4. Growth inhibitory effects of EGCG-CGA (4), EGC-CGA (5), and theaflavin 3,3'digallate (6).


18

Page 19 of 24


Table 1. 1H and 13C-NMR Data of 4 and 5a Position

4 1

H

5 13

C

1

H

13

C

δ (ppm), J (Hz)

δ (ppm)

δ (ppm), J (Hz)

δ (ppm)

79.1 67.5 25.5

4.93 (1H, s) 5.37 (1H, brs) 2.83 (1H, d, 16.8) 2.96 (1H, dd, 4.8, 16.8)

81.5 66.9 30.3

3”

5.10 (1H, s) 5.77 (1H, d, 4.1) 3.13 (1H, dd, 4.8, 17.4) 2.90 (1H, d, 17.0) 6.02 (1H, d, 2.0) 6.19 (1H, d, 1.8) 7.33 (1H, s) 8.06 (1H, s) 7.53 (1H, s) 8.27 (1H, d, 15.5) 6.18 (1H, d, 15.6) 1.94 (1H, d, 14.4) 2.27 (1H, dd, 14.8, 3.1) 4.07 (1H, m)

4” 5” 6” 7” G0 G1 G2 G3 G4 G5 G6

3.77 (1H, dd, 13.0, 3.0) 5.47 (1H, m) 2.17 (2H, d, 8.8) 6.74 (1H, s) 6.74 (1H, s)

74.3 71.8 39.6 168.2 168.0 122.1 109.0 147.0 140.5 147.0 109.0

2 3 4 4a 5 6 7 8 8a a b c d e f g h i j k 1’ 2’ 3’ 1” 2”

a

100.3 157.5 96.6 159.4 94.8 158.7 184.7 157.1 118.0 135.3 126.6 129.5 121.4 147.9 155.2 122.9 123.6 142.5 120.6 169.7 76.6 37.8 72.0

5.96 (1H, d, 1.8) 6.05 (1H, d, 2.4)

7.40 (1H, s) 8.05 (1H, s) 7.64 (1H, s)

8.39 (1H, d, 15.6) 6.34 (1H, d, 15.6)

2.00 (1H, m) 2.21 (1H, m) 4.3 (1H, s) 3.68 (1H, dd, 2.4, 9.0) 4.11 (1H, s) 2.13 (2H, m)

99.9 158.0 97.1 158.3 96.3 156.9 185.6 156.0 119.0 136.1 129.1 131.1 122.1 147.0 154.1 122.8 129.1 145.1 119.0 168.3 77.0 38.6 72.9 74.0 72.2 40.1

Recorded in CD3OD; s: singlet; d: doublet; m: multiplet; and brs: broad singlet.


19


Page 20 of 24

Figure 1.


20

Page 21 of 24


Figure 2.


21


m/z 779 [M-H]-

4:

Black tea

Black tea 50

561.04

435.09

100

100

50

MS3: 627/779 [M-H]-

4:

627.06

28.2

Black tea

761.19

489.20

50

609.24

191.04 0

0 100

28.3

0 100

627.14

100

Std

Std

50

50

561.11

50 761.23

0

10

5:

20 30 Time (min)

Relative Abundance

600

609.21

800

200

400 m/z

m/z

100

Black tea 50

27.5

Std 50

435.08

609.13

r.t.= 26.3 417.08

50 0 100

600

MS2: 627 [M-H]-

5:

27.5 26.3

0 100

489.17

Std

0 400

40

m/z 627 [M-H]-

100

435.16

191.04

0

0

B

MS2: 779 [M-H]-

4:

Relative Abundance

A

Relative Abundance

100

Page 22 of 24

481.12

435.05

r.t.= 27.5 417.10

50 0 100

489.08

609.17

435.10 489.14

Std 50

417.09

609.18

191.01

0 0

10

20 30 Time (min)

40

0

200

400 m/z

600

Figure 3.


22

Page 23 of 24


Figure 4.


23


Page 24 of 24

Table of Contents Graphic


24

Novel Theaflavin-Type Chlorogenic Acid Derivatives Identified in

Recommend Documents