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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

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Journal of Agricultural and Food Chemistry

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Novel Theaflavin-type Chlorogenic Acid Derivatives Identified in Black Tea

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

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Sang†, *

4 †

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

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

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

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‡

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

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§

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

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

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or [email protected])

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ACS Paragon Plus Environment

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ABSTRACT

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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provide more understanding of the complexity of black tea chemistry.

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

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INTRODUCTION

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

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

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

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

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

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

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remarkable bioactive potential.

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

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

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

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

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

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

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

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formation.14-18

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

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

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

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

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

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

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


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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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MATERIALS AND METHODS

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

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

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

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

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

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


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

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

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

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

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purchased from local supermarkets.

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Synthesis of theaflavin-type compounds

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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Preparation of black tea extract


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

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

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vials for LC/MS analysis.

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LC/MS Analysis

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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monitoring (SIM) mode.

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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


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

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CA) was used for all the mass data.

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

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

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

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

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

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linearity with correlation greater than 0.995.

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Nuclear Magnetic Resonance (NMR)

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

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

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compounds. The carbon NMR spectra are proton decoupled.

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MTT Assay

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

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

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

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

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

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

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

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

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

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


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

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viable cells in the control.

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RESULTS AND DISCUSSION

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Synthesis and Structural Elucidation of Compound 4

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

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

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

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

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

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

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

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

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

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

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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,

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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

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of the flavan-3-ol unit, and its

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

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

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

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

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

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

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


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

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

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

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

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

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

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

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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

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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

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(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),

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

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

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

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

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Structural Elucidation of Compound 5

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C signals of 4 was based on the

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

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

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

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

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

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

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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


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

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

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Identification of 4 and 5 in black tea.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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


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

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

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

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

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

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theaflavin-type compounds 4 and 5.

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

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

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

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

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

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

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

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

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

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theaflavins.

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

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

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

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

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

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

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


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

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

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

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

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

245

understanding of the complexity of the black tea chemistry.

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ACKNOWLEDGEMENT

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

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

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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).


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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


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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 ... - ACS Publications

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