Identification and Quantification of Bioactive Compounds in

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New Analytical Methods

Identification and Quantification of Bioactive Compounds in Diaphragma juglandis Fructus by UHPLC-Q-Orbitrap HRMS and UHPLC-MS/MS Rongxia Liu, Ziyan Zhao, Shengjun Dai, Xin Che, and Wanhui Liu J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b06890 • Publication Date (Web): 04 Mar 2019 Downloaded from http://pubs.acs.org on March 5, 2019

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Journal of Agricultural and Food Chemistry

Identification and Quantification of Bioactive Compounds in Diaphragma juglandis Fructus by UHPLC-Q-Orbitrap HRMS and UHPLC-MS/MS Rongxia Liu1,*, Ziyan Zhao1, Shengjun Dai, Xin Che, Wanhui Liu School of Pharmacy, Key Laboratory of Molecular Pharmacology and Drug Evaluation (Yantai University), Ministry of Education, Collaborative Innovation Center of Advanced Drug Delivery System and Biotech Drugs in Universities of Shandong, Yantai University, Yantai 264005, China

*Corresponding author: Tel: +86-53-56706030 Fax: +86-53-56706066 Email: [email protected] ORCID: 0000-0003-0998-4993 1

These authors contributed equally to this paper.

Email: Ziyan Zhao: [email protected] ORCID: 0000-0002-8797-4363 Shengjun Dai: [email protected] Xin Che: [email protected] Wanhui Liu: [email protected]

ACS Paragon Plus Environment


1

ABSTRACT

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Diaphragma juglandis fructus is the dry wooden diaphragm inside walnuts and is a

3

byproduct in food processing of walnut kernels. The purpose of our research is to

4

enrich the information on compounds in Diaphragma juglandis fructus to further

5

discover and exploit its potential nutritional value. In this study, new

6

quali-quantitative analytical approaches were developed to identify and determine

7

bioactive compounds in Diaphragma juglandis fructus. Two-hundred compounds,

8

including hydrolysable tannins, flavonoids, phenolic acids and quinones, were

9

identified by UHPLC-Q-Orbitrap HRMS, more than 150 of which were firstly

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discovered in Diaphragma juglandis fructus. Among them, 21 major dietary

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polyphenols with health-promoting effects were successfully quantified using

12

UHPLC-MS/MS, with total contents of 2.88-6.18 mg/g. This successful

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characterization and quantification of bioactive compounds in Diaphragma juglandis

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fructus gives a better understanding of its potential nutritional value and supports its

15

efficient development and reuse instead of discarding it as agro-food waste.

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KEYWORDS: Diaphragma juglandis fructus, identification, quantification,

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bioactive compounds, UHPLC-Q-Orbitrap HRMS, UHPLC-MS/MS

18 19 20 21 22


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INTRODUCTION

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The walnut (Juglans regia L.) is widely consumed as a high-nutrient food

25

throughout the world. It is composed of essential unsaturated fatty acids, melatonin,

26

vitamins, and a large number of polyphenols1. Today, the edible portion of walnuts

27

(seeds or kernels) is usually processed into various foods such as candies, cakes, and

28

beverages. However, Diaphragma juglandis fructus, the dry wooden diaphragm

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inside walnuts, is usually directly discarded or burned as fuel in walnut food

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processing. Currently, research and development efforts on the effective use of

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agro-food wastes with potential nutritional value have gained worldwide popularity

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and importance2-4. Some studies have revealed that byproducts from food or

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agricultural processing, such as sour cherry pomace5, cashew nut shells6, and

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pomegranate leaves and peels7-8, are promising sources of natural antioxidants and

35

other bioactive compounds.

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Recent reports showed that Diaphragma juglandis fructus contains diverse bioactive

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components, including phenolic acids, flavonoids, saponins, quinones, alkaloids, and

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polysaccharides9-14. Among these, phenolic acids and flavonoids are known as

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dietary polyphenols, which are widely found in plants or plant-derived foods such as

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fruits, vegetables, grains, tea and wine15. Dietary polyphenols exhibit a variety of

41

activities, such as antioxidant, anti-inflammatory, anti-tumor and cardiovascular

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protective activities16-19. However, to date, there has neither been a systematic and

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comprehensive description of the chemical composition nor a simultaneous

44

quantitative analysis of multiple bioactive compounds, especially dietary



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polyphenols, in Diaphragma juglandis fructus. Therefore, it is very important to

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develop new analytical approaches for its qualitative and quantitative analysis.

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Today, high resolution mass spectrometry (HRMS), like ultra-high-performance

48

liquid

49

(UHPLC-Q-Orbitrap HRMS), has been successfully used for rapid identification of

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plant constituents and can provide exact MS and MS/MS information20-22.

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Additionally, ultra-high-performance liquid chromatography coupled with tandem

52

mass spectrometry (UHPLC-MS/MS) has become one of the most effective

53

techniques for the rapid and accurate quantification of plant constituents23-24.

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Therefore, the development and application of these new technologies have made it

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increasingly possible to study the bioactive compounds in food and plants.

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In this study, novel quali-quantitative analytical methods with UHPLC-Q-Orbitrap

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HRMS and UHPLC-MS/MS approaches were developed for rapid and systematic

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identification and quantification of the components in Diaphragma juglandis fructus.

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Systematic characterization and quantification of bioactive compounds will be

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beneficial for understanding the potential nutritional value of Diaphragma juglandis

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fructus and for exploiting its utility instead of discarding it as agro-food waste.

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MATERIALS AND METHODS

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Chemicals and reagents

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Quinic acid, gallic acid, neochlorogenic acid, chlorogenic acid, syringic acid,

65

(-)-epicatechin,

66

myricitrin, ellagic acid, phlorizin, naringenin and phloretin were purchased from

chromatography

coupled

(-)-epigallocatechin

with

hybrid

gallate,

quadrupole-Orbitrap

vanillin,


(-)-epicatechin

HRMS

gallate,

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Chengdu Purechem-Standard Co., Ltd. (Sichuan, China). Protocatehuic acid,

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protocatechualdehyde, p-hydroxybenzoic acid, methyl gallate, (+)-catechin, vanillic

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acid, ethyl gallate, taxifolin, quercitrin, quercetin, luteolin and kaempferide were

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purchased from Shanghai Aladdin Reagent Co., Ltd. (Shanghai, China). Caffeic acid,

71

p-coumaric acid, syringaldehyde, ferulic acid, hyperoside, isoquercitrin, luteoloside,

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astilbin and chrysin were purchased from National Institute for the Control of

73

Pharmaceutical and Biological Products (Beijing, China). Dihydrophaseic acid and

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taxifolin-3-O-arabinofuranoside were isolated and purified from Diaphragma

75

juglandis fructus in our laboratory. The purities of reference standards were

76

determined to be over 98% by HPLC-UV. The structures of the 37 reference

77

standards are given in Fig 1.

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HPLC-grade acetonitrile, methanol, dimethyl sulfoxide (DMSO) and acetic acid

79

were purchased from Merck (Darmstadt, Germany). Other HPLC-grade reagents

80

were obtained from Fisher Scientific (Fairlawn, NJ). Deionized water was prepared

81

using a Milli-Q system (Merck Millipore, USA).

82

Ten batches of Diaphragma juglandis fructus were collected from different regions

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of China in the year 2017. The samples denoted as XJ-1, XJ-2 and XJ-3 were

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collected from Wulumuqi, Shihezi and Aksu of Xinjiang (XJ) province, respectively,

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SD-1, SD-2 and SD-3 were collected from Yantai, Ji’nan and Heze of Shandong (SD)

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province, respectively, HB-1 and HB-2 were collected from Langfang and Baoding

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of Hebei (HB) province, respectively, and SX-1 and SX-2 were collected from Xi’an

88

and Shangluo of Shanxi (SX) province, respectively. Diaphragma juglandis fructus



89

were authenticated by Shengjun Dai, School of Pharmacy, Yantai University.

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Voucher specimens were deposited at School of Pharmacy, Yantai University.

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Preparation of standards solutions and samples

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Thirty-seven reference standards were accurately weighed and dissolved in DMSO

93

to prepare individual stock solutions. All stock solutions were completely dissolved

94

in the mixed standard working solutions at concentrations of 2.0-4.0 μg/mL for

95

qualitative analysis. Twenty-one major dietary polyphenols were chosen for further

96

quantitative analysis. For the construction of calibration plots, 21 standard stock

97

solutions were mixed and further diluted with 50% methanol to produce a series of

98

standard solutions at the concentration range of 1.0-2500.0 μg/mL. All solutions

99

were stored at 4 °C in a refrigerator before analysis.

100

Sample preparation for identification: 1.5 g dried Diaphragma juglandis fructus

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powder (40 mesh) was weighed accurately and extracted with a 10-fold volume of

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acetone/cyclohexane (2:1, v/v) under ultrasonic conditions at room temperature for

103

30 min. The extract was centrifuged at 13,000 rpm for 10 min, and then 10 mL of

104

supernatants was evaporated from the extract under reduced pressure at 40 °C. The

105

resulting residue was dissolved in 1 mL of methanol. The analytes were filtered

106

through a 0.22-μm membrane before use.

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Sample preparation for quantification: 0.1 g dried Diaphragma juglandis fructus

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powder (40 mesh) was weighed accurately and extracted with 20 mL of 50%

109

methanol under ultrasonic conditions at room temperature for 30 min. The solution

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was allowed to cool naturally and weighed again, and the lost weight was made up


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with 50% methanol and shaken. The solution was centrifuged at 13,000 rpm for 10

112

min. The supernatants were diluted 1:10 (v/v) with 50% methanol for the analysis of

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ellagic acid and methyl gallate, and a 1:1 dilution was used for analyzing the rest of

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the compounds. All samples were filtered (0.22-μm) before analyses.

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Chromatographic and mass spectrometric conditions

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Identification of components in Diaphragma juglandis fructus A Waters

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Acquity H-Class UPLC system (Waters, Milford, MA, USA) coupled with a Q

118

ExactiveTM Orbitrap MS system (Thermo Scientific, Waltham, MA, USA) was used

119

for the identification of components in Diaphragma juglandis fructus. An

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ACQUITY UPLC HSS T3 column (2.1 mm × 100 mm, 1.8 μm) was applied for

121

chromatographic separation with column temperature at 30 °C. The mobile phase

122

consisted of water containing 0.1% acetic acid as eluent A and acetonitrile/methanol

123

(4:1, v/v) as eluent B. The gradient elution program was as follows: 0-1 min, 4% B;

124

1-12 min, 4-32% B; 12-18 min, 32-60% B; 18-20 min, 60-95% B; 20-22 min, 95%

125

B. The flow rate was 0.4 mL/min and the injection volume was 3 μL.

126

The mass spectrometer was operated with the heated electrospray ionization source in

127

both positive and negative ion modes. The key parameters were as follows: spray

128

voltage, +3.8 kV/-2.8 kV; sheath gas flow rate, 35 arbitrary units (Arbs); auxiliary gas

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flow rate, 10 Arbs; sweep gas flow rate, 0 Arbs; capillary temperature, 325 °C;

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auxiliary gas heater temperature, 350 °C; scan modes, full MS with a resolution of

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70,000 FWHM (full width at half-maximum, at m/z 200) and data-dependent MS/MS

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with a resolution of 17,500 FWHM; stepped normalized collision energy, 20, 40 and



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60 eV and scan range, m/z 80-1,200. Data acquisition and processing were carried out

134

with Xcalibur 4.1 and Mass frontier 7.0 software (Thermo Scientific), respectively.

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Quantification of bioactive compounds in Diaphragma juglandis fructus A

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Shimadzu LC-30AD system (Shimadzu Corporation, Kyoto, Japan) coupled with a

137

SCIEX Triple QuadTM 4500 system (SCIEX, Foster City, CA, USA), was used for

138

the determination of bioactive compounds. The column, column temperature, mobile

139

phase and flow rate were the same as described in the section of “Identification of

140

components in Diaphragma juglandis fructus”. The gradient program was as

141

follows: 4% B from 0 to 1 min, 4-35% B from 1 to 15 min, 35-95% B from 15 to 18

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min, 95% B from 18 to 20 min. The injection volume was 2 μL.

143

The mass spectrometer was operated with the electrospray ionization source in

144

negative ion mode. Analytes were detected in multiple reaction monitoring (MRM)

145

mode, and the optimized parameters are listed in Table S1 (Supporting information).

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The other major parameters used were as follows: ion spray voltage, -4,500 V; source

147

temperature, 550 °C; curtain gas, 35 psi; ion source gas 1, 55 psi; ion source gas 2, 55

148

psi; entrance potential, -10 V; collision gas, 7 psi. Analyst 1.6.3 and MultiQuant 3.0.2

149

software (SCIEX) were used to acquire and analyze the experimental data,

150

respectively.

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RESULTS AND DISCUSSION

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Comparison with prior research on analytical methods

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Many different analytical methods have been used for the identification and

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quantification of polyphenols in recent years. LC-Q-TOF is most commonly


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employed for identification while Q-Orbitrap has become an increasingly popular

156

identification option25. For quantification, LC with photodiode array detection

157

(LC-DAD) and LC-QqQ-MS/MS are extensively utilized26-27.

158

In our study, the advanced UHPLC-Q-Orbitrap HRMS technique was used for the

159

characterization of components in Diaphragma juglandis fructus. Compared to

160

traditional Q-TOF, Q-Orbitrap exhibits a significantly higher mass-resolving power

161

with a resolution as high as 140,000 FWHM. Furthermore, previous studies have

162

shown that Q-Orbitrap allows for powerful mass-axis calibration, which can remain

163

stable for weeks and are hardly affected by environmental influences20. Orbitrap can

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also be used to identify compounds at trace level concentrations22. In short, Orbitrap

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has a strong capacity for the identification of compounds in sample mixtures, and it

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allows for highly rapid screening analysis with relatively simple sample preparation.

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The UHPLC-MS/MS technique was chosen for our study to determine polyphenols

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in Diaphragma juglandis fructus. The developed LC-MS/MS method was much

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better than LC-DAD method which has been extensively used for quantification of

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phenolic compounds in walnut kernels or other food28-31. Firstly, the developed

171

LC-MS/MS method had better sensitivity since it had lower limit of detection (LOD)

172

and limit of quantification (LOQ), even the LOD value of some compounds was as

173

low as 0.01 ng/mL. Thus, the developed LC-MS/MS method had a great advantage

174

for the determination of trace compounds with LOD of ng/mL in sample matrix.

175

Additionally, the developed LC-MS/MS method possessed a higher linear dynamic

176

range, which was suitable for quantitative analysis of compounds with large



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discrepancy in level of content in different samples. Another benefit of LC-MS/MS

178

is that MRM mode is available in MS/MS, allowing for the simultaneously quantify

179

more components with shorter analytical time. Moreover, in our knowledge, this is

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the first LC-MS/MS method for the quantification of phenolic compounds in

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Diaphragma juglandis fructus. Compared to the previous LC-MS/MS method of

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phenolic compounds in walnut kernels32, our method quantified more compounds

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with higher sensitivity. Therefore, UHPLC-Q-Orbitrap HRMS with its robust

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qualitative ability and UHPLC-MS/MS which is obviously superior for quantitative

185

research were selected for use in this study.

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An integrated identification strategy for the components in Diaphragma

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juglandis fructus

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In this study, we proposed a three-step integrated strategy (Fig 2) for systematic

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characterization and identification of components in Diaphragma juglandis fructus.

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Firstly, a compound library including reference compounds, components of Juglans

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and other types of compounds of interest was built according to pre-experiments, a

192

large number of literature references, and online databases such as MassBank

193

(https://massbank.eu/MassBank/),

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ChemSpider

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(http://tcm.cmu.edu.tw/). This compound library contains compound name, formula,

196

structure, and fragment information for each compound. Secondly, the discovery and

197

identification of compounds in Diaphragma juglandis fructus were processed

198

successively as follows: high throughput search by matching with library, manual

mzCloud

(http://www.chemspider.com/)

(https://www.mzcloud.org/), and


TCM

Database@Taiwan

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search for trace compounds and fragment ion search (FISh) for compounds with

200

common fragments, and finally, fragmentation pathways were elucidated by exact

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MS and MS/MS data and fragment information in the library. Diagnostic product

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ions (DPIs) and neutral loss were not only used to elucidate the fragmentation and

203

mechanisms of compounds, but also to “FISh” for compounds with similar structures.

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Based on this strategy, 200 compounds were successfully identified, more than 150

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of which were firstly discovered in Diaphragma juglandis fructus. Among the

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identified 200 components, 37 of them were unambiguously identified with

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reference standards. Information on all compounds identified in this study is

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summarized in Table 1. Total ion chromatograms in positive and negative modes of

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Diaphragma juglandis fructus extracts are shown in Fig 3.

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Characterization of components in Diaphragma juglandis fructus

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To acquire more abundant information on compounds present in Diaphragma

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juglandis fructus, extraction solvents of a wide polarity range, including methanol,

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95% ethanol, acetone and cyclohexane were investigated. Among them, acetone

214

showed better extraction efficiency with less interference. Furthermore, different

215

proportions of acetone/water and of acetone/cyclohexane were tested. As a result,

216

acetone/cyclohexane (2:1) was selected as the extraction solvent for identification

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since it showed more chromatographic peaks and less interference (Fig S1).

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Identification of hydrolysable tannins Hydrolysable tannins are divided into

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ellagitannins and gallotannins15. Ellagitannins, as esters of hexahydroxydiphenolic

220

acid (HHDP) and monosaccharide, are dehydrated after hydrolysis and then



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converted by spontaneous lactonization into ellagic acid33-34. Compounds 3, 4 and 5

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all displayed [M-H]- ions at m/z 481, and their intense fragment ion at m/z 301

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[M-H-C6H12O6]- indicated the loss of a glucose and the existence of an ellagic acid

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group. Therefore,

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HHDP-glucose isomers34-36. Similarly, a series of compounds with common

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fragments were identified by “FISh” with DPI at m/z 301, such as compounds 9, 14,

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35, 48 and 66. Gallotannins are another type of hydrolysable tannin present in

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nature37-38. Compounds 2, 6, 7, 11, 15, 167 and 170 all gave the same [M-H]- ions at

229

m/z 331 with product ions at m/z 271 [M-H-C2H4O2]-, 211 [M-H-C4H8O4], 169

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[M-H-C10H6O5]- and 125 [M-H- C10H6O5-CO2]-. The DPIs at m/z 169 and 125

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confirmed the existence of a galloyl group. Thus, these compounds were tentatively

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identified as monogalloyl-glucose isomers34. Similarly, digalloyl-glucose (10, 19, 23,

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27, 30, 44, 50 and 52), trigalloyl-glucose (59, 72, 94, 98, 100 and 116) and

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tetragalloyl-glucose (131, 137 and 143) isomers were also identified in Diaphragma

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juglandis fructus extracts34,37. MS/MS spectra of representative ellagitannins and

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gallotannins are shown in Fig 4A and 4B, respectively.

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Identification of phenolic acids Phenolic acids are prone to cleavage of CO2 from

238

the carboxylic acid moiety in negative ion mode39. Neutral loss of CO2 was detected

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in the MS/MS data of ellagic acid (152), gallic acid (8), protocatehuic acid (20),

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p-hydroxybenzoic acid (37), vanillic acid (61), caffeic acid (65), syringic acid (76)

241

and p-coumaric acid (117). Compounds 24 and 43 showed the same molecular ions

242

at m/z 353 with DPIs at m/z 191 and 179. As a pair of isomers, these two

these three

compounds were


tentatively

identified

as

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compounds were confirmed as neochlorogenic acid and chlorogenic acid by

244

comparison to the reference standards.

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Identification of quinones The fragmentation of quinones is generally caused by

246

the cleavage of the substituents or the elimination of carbon on the benzene ring,

247

such as the cleavage of the hydroxy group on the benzene ring leading to the neutral

248

loss of H2O or CO39. In this study, quinones were identified by characteristic ions

249

produced by the continuous loss of H2O, CO and/or CH2. For example, compounds

250

12, 45, 74, 85 and 141 were tentatively identified as naphthalenediol isomers40 based

251

on the molecular ions at m/z 161 [M+H]+ and fragment ions at m/z 133 [M+H-CO]+,

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115 [M+H-CO-H2O]+ , 105 [M+H-2CO]+ and 91 [M+H-2CO-CH2]+.

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The fragmentation pathways of catechin and quercetin derivatives were also

254

discussed and are included in the Supporting Information.

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Selection of dietary polyphenols and optimization of experimental conditions

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for quantification

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Among the 200 compounds identified in Diaphragma juglandis fructus, dietary

258

polyphenols represented the majority of components, including hydrolysable tannins,

259

flavonoids and phenolic acids. Notably, dietary polyphenols as bioactive ingredients

260

have been proven to be beneficial to the human body15-19,41. Thus, 21 major dietary

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polyphenols were chosen for further quantification in Diaphragma juglandis fructus

262

in this study, and the amounts of these dietary polyphenols in Diaphragma juglandis

263

fructus were compared with walnuts and other foods.

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Sample preparation is critical for the development of quantitative methods. In order



265

to optimize the sample preparation conditions, the extraction solvent (25%, 50%,

266

75%, 100% methanol), solid-liquid ratio (1:50, 1:100, 1:200, 1:300), and extraction

267

time (5, 15, 30, 60 min) were examined. Based on the sum of the peak areas of all

268

analytes, the extraction efficiency was evaluated to obtain the best sample

269

preparation method. The final optimized result was that 0.1 g Diaphragma juglandis

270

fructus powder was extracted with 20 mL 50% methanol under ultrasonic conditions

271

at room temperature for 30 min.

272

For UHPLC-MS/MS conditions, the type of column, elution condition and injection

273

volume were optimized. The ACQUITY UPLC HSS T3 column was selected with

274

better peaks shapes and improved analyte responses. The analysis process used

275

gradient elution to simultaneously separate 21 compounds in 20 min, which gave

276

better separation for those compounds. To obtain the maximum response of each

277

compound, the precursor ion (Q1), product ion (Q3), declustering potential (DP) and

278

collision energy (CE) were optimized.

279

Method developed for quantification of dietary polyphenols in Diaphragma

280

juglandis fructus was verified by linearity, LOD, LOQ, precision, repeatability,

281

stability and recovery. The detailed method validation process is shown in the

282

Supporting Information, and results are shown in Tables S2 and S3.

283

Application to dietary polyphenols measurement in Diaphragma juglandis

284

fructus

285

The newly developed and validated LC-MS/MS method was applied to

286

simultaneously quantify 21 dietary polyphenols in 10 batches of Diaphragma


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juglandis fructus acquired from different regions in China, and the results of

288

quantification are displayed in Table 2. The total amount of all compounds in each

289

batch varied from 2.88 to 6.18 mg/g.

290

Ellagic acid was the most abundant phenolic compound found in Diaphragma

291

juglandis fructus samples in this study, ranging from 518.38 to 1733.64 μg/g.

292

Previous studies reported that ellagic acid was a major bioactive phenolic compound

293

in walnut kernels28,32. The content of ellagic acid in black walnut kernels and English

294

walnut kernels ranged from 11.35 to 72.05 μg/g and from 217.3 to 704.7 μg/g,

295

respectively28,32. Our results indicated that the content of ellagic acid in Diaphragma

296

juglandis fructus was similar to or even higher than that in walnut kernel. In addition,

297

according to the Phenol-Explorer database (http://phenol-explorer.eu/), different

298

levels of ellagic acid have been found in blackberry (436.70 μg/g), cloudberry

299

(153.00 μg/g), strawberry (12.40 μg/g) and red raspberry (21.20 μg/g), as well in as

300

fruit juices, such as muscadine grape juice (0.90 mg/100 mL). It has also been found

301

in brandy (1.13 mg/100 mL) and whisky (0.82 mg/100 mL). It was not difficult to

302

conclude that the content of ellagic acid in Diaphragma juglandis fructus

303

(518.38-1733.64 μg/g) was considerable since it was similar to or even higher than

304

that in many foods. The amount of gallic acid in Diaphragma juglandis fructus

305

(89.87-219.09 μg/g) determined in this study was also much higher than that

306

reported in walnut kernels (5.33-9.57 μg/g)29. Gallic acid, a common dietary

307

component, has been commonly found in various foods, such as bananas (10.00

308

μg/g), eggplants (1.40 μg/g), cauliflower (6.90 μg/g) and olives (0.20 μg/g), as



309

reported by the Phenol-Explorer database. Previous studies also reported that

310

syringic acid was the most abundant phenolic acid in walnut kernels with an average

311

content of 288.75 μg/g or 338.29 μg/g28,30, which is higher than that in Diaphragma

312

juglandis fructus (11.44-26.76 μg/g).

313

As a type of well-known dietary polyphenol, catechins are the main bioactive

314

ingredients in green tea42-44. Diaphragma juglandis fructus have also been used as an

315

herbal tea and a dietary supplement in some areas45. Catechins including

316

(+)-catechin, (-)-epicatechin, (-)-epicatechin gallate and (-)-epigallocatechin gallate

317

were found in the extracts of Diaphragma juglandis fructus in this study. Their

318

amounts were (+)-catechin (251.69-693.32 μg/g) > (-)-epicatechin gallate

319

(22.30-194.79 μg/g) > (-)-epicatechin (8.82-36.44 μg/g) > (-)-epigallocatechin

320

gallate (0.43-2.21 μg/g). By comparison, the content of (+)-catechin was within or

321

lower than the range of (+)-catechin in green tea (1.20-2820.00 μg/g)43. However,

322

the contents of (+)-catechin, (-)-epicatechin and (-)-epicatechin gallate in

323

Diaphragma juglandis fructus were much higher than that in walnut kernels

324

(14.80-82.00 μg/g, 0.34-1.49 μg/g and 2.72-13.22 μg/g, respectively)28,32. These

325

results indicated that the amount of catechins in Diaphragma juglandis fructus was

326

also considerable.

327

As common bioactive polyphenols, quercetin derivatives are widespread in human

328

diets via fruits and vegetables46. In this study, quercitrin, isoquercitrin and taxifolin

329

were found in the ranges of 145.42-983.58 μg/g, 20.89-116.68 μg/g and

330

33.99-153.31 μg/g, respectively. Notably, the content of isoquercitrin in Diaphragma


Page 16 of 48

Page 17 of 48


331

juglandis fructus was higher than that reported in walnut kernels (1.02-4.06 μg/g)32

332

and many other foods, such as whole apples (5.40 μg/g), pears (2.10 μg/g), broccoli

333

(18.00 μg/g), and red onions (18.00 μg/g). Taxifolin-3-O-arabinofuranoside also

334

showed a significant concentration in Diaphragma juglandis fructus, varying from

335

519.60 to 2181.84 μg/g, while its content has not been reported in walnut kernels.

336

Our results indicated that Diaphragma juglandis fructus was rich in dietary

337

polyphenols, and the amounts of many phenolic compounds were similar to or even

338

higher than that in walnut kernels and various fruits, vegetables and beverages.

339

Numerous studies have shown that dietary polyphenols exhibit various activities in

340

the human body. Antioxidant activity is one of the most known bioactivities of

341

polyphenolic compounds, and regular consumption of antioxidant-rich foods helps to

342

eliminate free radicals in the body and improve antioxidant status, thereby

343

preventing cardiovascular diseases47. Moreover, epidemiological and clinical

344

research has demonstrated that an intake of large amounts of dietary polyphenols can

345

effectively prevent chronic diseases48-50, and dietary polyphenols may be effective in

346

dietary intervention for chronic disease management51. Thus, since it is rich in

347

dietary polyphenols with health-promoting effects, Diaphragma juglandis fructus

348

has great potential to be developed for functional foods and nutraceuticals instead of

349

being relegated to agro-food waste. Further investigations on Diaphragma juglandis

350

fructus should be carried out, for example, studies on quantification, bioactivity,

351

bioavailability and toxicology of other types of components in Diaphragma

352

juglandis fructus and their interactions with dietary polyphenols. The development



353

of nutritional and functional foods is undoubtedly a critical and innovative part of

354

the overall food market.

355

In summary, new quali-quantitative approaches were developed to screen and

356

quantify bioactive compounds in Diaphragma juglandis fructus, which displayed

357

accurate, sensitive, economic and time-saving advantages. The successful

358

characterization of 200 components and quantification of 21 major dietary

359

polyphenols in Diaphragma juglandis fructus will not only benefit exploiting its

360

hidden nutritional value but also support its efficient development and reuse. Based

361

on comprehensive composition identification and accurate quantification strategy,

362

the developed methods could also be applied to other agro-food wastes for rapid

363

identification and quantification of bioactive constituents.

364

Author Contributions

365

RL and WL conceived and designed the study. ZZ performed experiments and

366

analyzed data. RL, SD and XC analyzed data and supported planning and

367

interpretation of experiments. RL and ZZ drafted the manuscript. All authors revised

368

and approved the final manuscript.

369

Conflict of Interest The authors declare that they have no conflict of interest.

370

Acknowledgements

371

This work was funded by the National Natural Science Foundation of China (No.

372

81603326) and supported by Taishan Scholar Project.

373

Supporting Information LC-UV-MS Chromatograms of different extraction

374

solvents for identification (Fig S1). Identification of catechin derivatives and


Page 18 of 48

Page 19 of 48


375

quercetin derivatives in Diaphragma juglandis fructus; Method validation for

376

quantification of dietary polyphenols in Diaphragma juglandis fructus; Optimized

377

multiple-reaction-monitoring parameters for 21 reference standards (Table S1);

378

Calibration curve, linear range, LOD and LOQ for 21 analytes (Table S2); Precision,

379

repeatability, stability and recovery of 21 analytes (Table S3); MS/MS spectra of

380

catechin derivatives (Figure S2); MS/MS spectra of quercetin derivatives (Figure S3);



381

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FIGURE CAPTIONS Figure 1. Chemical structures of 37 reference standards in Diaphragma juglandis fructus. (Glc: glucosyl; Rha: rhamnosyl; Ara: arabinosyl; Gal: galactosyl; G: galloyl; Caff: caffeoyl) Figure 2. A proposed integrated strategy for identification of the components in Diaphragma juglandis fructus. Figure 3. Total ion chromatograms in positive and negative mode of Diaphragma juglandis fructus extracts (representative components are labeled). Figure 4. MS/MS spectra of hydrolysable tannins: (A) compound 3 (HHDP-glucose isomer), DPI at m/z 301; (B) compound 2 (monogalloyl-glucose isomer), DPIs at m/z 169 and 125. Figure 5. Typical MRM chromatogram of mixed reference standards (21 analytes). Peak identification: C8, gallic acid; C20, protocatehuic acid; C24, neochlorogenic acid; C33, protocatechualdehyde; C42, methyl gallate; C56, (+)-catechin; C61, vanillic acid; C76, syringic acid; C93, (-)-epicatechin; C95, (-)-epigallocatechin gallate; C146, (-)-epicatechin gallate; C148, taxifolin; C149, myricitrin; C151, hyperoside; C152, ellagic acid; C155, isoquercitrin; C157, astilbin; C159, taxifolin-3-O-arabinofuranoside; C176, quercitrin; C185, phlorizin; C192, quercetin.



Page 30 of 48

Table 1. Characterization of the components from Diaphragma juglandis fructus extracts by UHPLC-Q-Orbitrap HRMS NO.

tR (min)

Formula

Ion

Measured

mode

Mass (m/z)

MS/MS Fragments (m/z)

Error (ppm)

Compound Identification

1a

0.67

C7H12O6

-

191.05542

117.04 (10), 93.03 (18), 85.03 (60)

-3.61

Quinic acid

2

0.87

C13H16O10

-

331.06766

211.02 (18), 169.01 (65), 125.02 (100)

1.78

Monogalloyl-glucose

3

1.02

C20H18O14

-

481.06226

301.00 (100), 275.02 (54), 257.01 (24), 229.01 (35)

-0.25

HHDP-glucose isomer

4

1.14

C20H18O14

-

481.06253

301.00 (100), 275.02 (48), 257.01 (17), 229.01 (26)

0.31

HHDP-glucose isomer

5

1.29

C20H18O14

-

481.06265

301.00 (100), 275.02 (60), 257.01 (28), 229.01 (38)

0.56

HHDP-glucose isomer

0.76

Monogalloyl-glucose

6 7 8

a

271.08 (2), 211.02 (2), 169.01 (6), 125.02 (9),

1.46

C13H16O10

-

331.06732

1.78

C13H16O10

-

331.06717

271.05 (57), 211.02 (100), 169.01 (60), 125.02 (80),

0.30

Monogalloyl-glucose

1.87

C7H6O5

-

169.01335

125.02 (100), 107.01 (1)

-5.32

Gallic acid

-0.63

Galloyl-HHDP-glucose

-4.10

Digalloyl-glucose

108.02 (100)

301.00 (100), 275.02 (33), 257.01 (14), 229.01

9

1.94

C27H22O18

-

633.07294

10

2.20

C20H20O14

-

483.07605

11

2.30

C13H16O10

-

331.06689

271.05 (48), 211.02 (100), 169.01 (79), 125.02 (79)

-0.54

Monogalloyl-glucose

12

2.95

C10H8O2

+

161.05940

133.06 (100), 115.05 (17), 105.07 (62), 91.05 (6)

-1.92

Naphthalenediol isomer

13

2.99

C27H22O18

-

633.07104

-3.63


14

3.17

C34H24O22

-

783.06780

301.00 (100), 275.02 (85), 257.01 (32), 229.01 (34)

-1.09

bis-HHDP-glucose

15

3.28

C13H16O10

-

331.06686

169.01 (54), 125.02 (100)

-0.63

Monogalloyl-glucose

16

3.31

C8H8O4

-

167.03418

139.04 (26), 123.04 (100), 109.03 (17)

-4.79

Hydroxymandelic acid

17

3.36

C16H18O8

+

339.10740

185.04 (1), 147.04 (1)

-0.12

Trihydroxynaphthaline glucoside

18

3.49

C45H38O18

-

865.19800

407.08 (9), 289.07 (9), 245.08 (7), 151.04 (14),

-0.62

Procyanidin trimer

(25), 169.01 (8), 125.02 (26) 331.07 (4), 313.06 (3), 211.02 (4), 169.01 (50), 125.02 (100)

301.00 (90), 275.02 (50), 257.01 (25), 229.02 (31), 169.01 (9), 125.02 (27)


Page 31 of 48


125.02 (100) 19

3.74

C20H20O14

-

483.07794

20 a

3.81

C7H6O4

-

153.01842

21

3.87

C34H24O22

-

22

4.06

C14H18O10

23

4.31 4.44

25

331.07 (5), 313.06 (4), 211.02 (3), 169.01 (67),

-0.19

Digalloyl-glucose

109.03 (100), 81.03 (2)

-5.95

Protocatehuic acid

783.07050

301.00 (100), 275.02 (28), 257.01 (13), 229.01 (25)

2.36

bis-HHDP-glucose

-

345.08240

168.01 (9), 124.02 (5)

-0.93

Methyl galloyl hexoside

C20H20O14

-

483.07751

169.01 (58), 125.02 (100)

-1.08

Digalloyl-glucose

C16H18O9

-

353.08768

191.06 (89), 179.03 (25), 135.04 (100)

-0.37

Neochlorogenic acid

4.46

C15H20O10

-

359.09837

0.00

Dimethyl galloyl hexoside

26

4.47

C30H26O13

-

593.12982

407.08 (26), 289.07 (15), 151.04 (34), 125.02 (100)

-0.40

(Epi)gallocatechin-(epi)catechin

27

4.66

C20H20O14

-

483.07916

313.06 (14), 169.01 (73), 125.02 (100)

2.34

Digalloyl-glucose

28

4.70

C8H8O3

-

151.03973

136.02 (78), 123.04 (100), 108.02 (15)

-2.25

Vanillin isomer

29

4.74

C8H8O5

-

183.02913

168.01 (54), 152.98 (35), 124.02 (100)

-4.21

Methyl gallate isomer

30

4.82

C20H20O14

-

483.07870

1.39

Digalloyl-glucose

31

5.02

C16H18O8

+

339.10590

-4.54


32

5.04

C27H22O18

-

633.07368

0.54


33 a

5.25

C7H6O3

-

137.02336

108.02 (5), 93.03 (1)

-7.74

Protocatechualdehyde

34

5.37

C34H24O22

-

783.06780

301.00 (100), 275.02 (35), 257.01 (17), 229.01 (27)

-1.09

bis-HHDP-glucose

35

5.37

C41H28O27

-

951.07360

301.00 (100), 275.02 (41), 257.01 (17), 229.01 (29)

-0.97

Trigalloyl -HHDP-glucose

36

5.40

C27H22O18

-

633.07483

2.35


37 a

5.48

C7H6O3

-

137.02332

93.03 (100)

-8.03

p-Hydroxybenzoic acid

38

5.54

C16H18O8

-

337.09283

173.04 (2), 163.04 (60), 119.05 (100), 93.03 (7)

-0.18

Coumaroylquinic acid

39

5.70

C30H26O12

-

577.13470

425.09 (6), 407.08 (22), 289.07 (25), 245.08 (11),

-0.78

Procyanidin dimer

24 a

125.02 (100)

197.05 (27), 182.02 (48), 167.00 (26), 153.05 (12), 138.03 (82), 123.01 (100)

313.06 (5), 271.05 (15), 211.02 (59), 169.01 (74), 125.02 (100) 277.03 (8), 231.03 (2), 177.05 (33) 301.00 (100), 275.02 (18), 257.01 (10), 229.01 (15), 169.01 (7), 125.02 (17)

481.06 (2), 301.00 (100), 275.02 (26), 257.01 (12), 229.01 (17), 169.01 (5), 125.02 (12)



Page 32 of 48

151.04 (25), 137.02 (30), 125.02 (100), 109.03 (44) 40

5.72

C10H10O4

-

193.04982

175.04 (100), 147.04 (18), 131.00 (32), 119.05 (26)

-4.20

Ferulic acid isomer

41

5.87

C16H20O9

-

355.10333

175.04 (100), 147.04 (7), 119.05 (3)

-0.37

Juglanoside D isomer

5.87

C8H8O5

-

183.02916

168.01 (22), 124.02 (18), 106.00 (2)

-4.04

Methyl gallate

5.89

C16H18O9

-

353.08759

-0.62

Chlorogenic acid

44

5.92

C20H20O14

-

483.07797

271.04 (13), 211.02 (37), 169.01 (74), 125.02 (100)

-0.12

Digalloyl-glucose

45

5.93

C10H8O2

+

161.05942

133.06 (61), 115.05 (18), 105.07 (24), 91.05 (4)

-1.80


-3.62

Procyanidin dimer

-4.79

Hydroxyphenyl-propionic acid

-0.41

Digalloyl-HHDP-glucose

-0.65

Coumaric acid hexoside isomer

-1.57

Digalloyl-glucose

-0.65

Epicatechin-3-O-glucoside isomer

-0.93

Digalloyl-glucose

-0.76

Procyanidin trimer

42 a 43

a

425.09 (5), 407.08 (22), 289.07 (21), 245.08 (15),

46

6.00

C30H26O12

-

577.13306

47

6.01

C9H10O3

-

165.05493

48

6.09

C34H26O22

-

785.08398

49

6.09

C15H18O8

-

325.09268

50

6.15

C20H20O14

-

483.07727

51

6.15

C21H22O12

-

465.10355

52

6.23

C20H20O14

-

483.07758

53

6.24

C45H38O18

-

865.19788

54

6.26

C14H10O9

-

321.02509

169.01 (57), 125.02 (100)

-0.37

Digallate

55

6.27

C10H10O3

-

177.05487

159.04 (39), 133.06 (51), 131.05 (7)

-4.80

Isosclerone isomer

-1.83

(+)-Catechin

56 a

6.31

C15H14O6

-

289.07123

57

6.42

C21H22O13

-

481.09787

151.04 (22), 137.02 (30), 125.02 (100), 109.03 (37) 135.04 (100), 108.02 (8), 93.03 (32) 301.00 (100), 275.02 (44), 257.01 (17), 229.01 (20), 169.01 (12), 125.02 (25) 163.04 (21), 119.05 (100), 93.03 (13) 313.06 (6), 271.05 (19), 211.02 (52), 169.01 (72), 125.02 (100) 303.05 (11), 285.04 (16), 179.00 (15), 151.00 (31), 125.02 (100), 107.01 (11) 331.07 (4), 313.06 (5), 211.03 (3), 169.01 (64), 125.02 (100) 577.14 (3), 407.08 (12), 289.07 (12), 245.08 (6), 151.04 (17), 125.02 (100)

245.08 (15), 203.07 (27), 151.04 (29), 137.02 (24), 125.02 (36), 123.04 (87), 109.03 (100), 97.03 (29) 271.05 (11), 211.02 (39), 169.01 (80), 151.01 (26), 125.02 (100)


-1.85

Galloyl dexoyhexoside isomer

methylgalloyl

Page 33 of 48


58

6.43

C22H18O11

-

457.07745

303.05 (19), 169.01 (10), 151.04 (100), 125.02 (51)

-0.39

Epigallocatechin gallate isomer

59

6.50

C27H24O18

-

635.08860

313.06 (24), 169.01 (76), 125.02 (100)

-0.61

Trigalloyl-glucose

60

6.52

C45H38O18

-

865.19775

-0.91

Procyanidin trimer

61 a

6.58

C8H8O4

-

167.03435

-3.77

Vanillic acid

62

6.63

C27H22O18

-

633.07062

-4.30


63

6.63

C10H8O3

-

175.03941

-3.77

7-Hydroxy-methylcoumarin

-2.32

Procyanidin trimer

-4.64

Caffeic acid

-0.75

Galloyl-bis-HHDP-glucose

-1.09

Procyanidin dimer

-0.74


-0.79

Procyanidin pentamer

64

6.67

C45H38O18

-

865.19653

65 a

6.82

C9H8O4

-

179.03415

66

6.87

C41H28O26

-

935.07890

67

6.87

C30H26O12

-

577.13452

68

6.91

C15H18O8

-

325.09265

69

6.97

C75H62O30

-

70

7.07

C10H10O4

71

7.07

C16H20O9

72

7.09

73 74 75

577.14 (2), 425.09 (2), 407.08 (10), 289.07 (9), 245.08 (7), 151.04 (15), 125.02 (100) 152.01 (100), 123.04 (21), 108.02 (82) 301.00 (100), 275.02 (19), 257.01 (11), 229.01 (15), 125.02 (7) 147.04 (27), 131.05 (59) 425.09(4), 407.08 (10), 289.07 (9), 245.08 (7), 161.02 (37), 125.02 (100) 135.04 (100), 107.05 (3) 301.00 (92), 275.02 (100), 257.01 (52), 229.01 (57), 125.02 (47) 425.09 (4), 407.08 (18), 289.07 (20), 245.08 (11), 151.04 (21), 137.02 (24), 125.02 (100) 265.07 (8), 235.06 (4), 205.05 (13), 163.04 (20), 119.05 (68)

720.15845

577.14 (1), 407.08 (11), 289.07 (14), 245.08 (7),

[M-2H]2-

151.04 (19), 125.02 (100)

-

193.04990

175.04 (100), 147.04 (9), 131.00 (2)

-3.78

Ferulic acid isomer

-

355.10321

175.04 (100), 147.04 (3)

-0.70


C27H24O18

-

635.09070

2.69

Trigalloyl-glucose

7.09

C10H8O3

-

175.03922

147.04 (14), 131.05 (18)

-4.86


7.10

C10H8O2

+

161.05942

133.06 (61), 115.05 (17), 105.07 (24), 91.05 (3)

-1.80


7.13

C30H26O12

-

577.13452

-1.09

Procyanidin dimer

313.06 (11), 271.05 (4), 211.02 (11), 169.01 (90), 125.02 (100)

425.09 (8), 407.08 (25), 289.07 (25), 245.08 (13), 151.04 (23), 137.02 (25), 125.02 (100), 109.03 (38)



76 a

7.15

C9H10O5

-

77

7.18

C75H62O30

-

78

7.22

C21H22O12

-

465.10336

79

7.25

C34H26O22

-

785.08301

80

7.29

C21H22O11

-

449.10864

81

7.35

C16H18O8

+

82

7.36

C16H18O8

83

7.46

C21H22O13

197.04488

182.02 (46), 167.00 (47), 153.05 (5), 138.03 (9), 123.01 (100)

720.15863

577.14 (2), 425.09 (2), 407.08 (9), 289.07 (19),

[M-2H]2-

245.08 (7), 151.04 (20), 125.02 (100) 303.05 (100), 285.04 (21), 273.04 (60), 243.03 (48), 201.02 (38), 175.04 (73) 301.00 (100), 275.02 (33), 257.01 (13), 229.01

Page 34 of 48

-3.40

Syringic acid

-0.54


-1.05

Quercetin-3-O-galactopyranoside isomer

-1.64


287.06 (18), 259.06 (88), 151.00 (41), 125.02 (100)

-0.65

Astilbin isomer

339.10730

177.05 (2), 147.04 (100), 119.05 (27)

-0.41


-

337.09290

173.04 (95), 163.04 (20), 119.05 (42), 93.03 (100)

0.03

Coumaroylquinic acid Vanillic

-

481.09839

313.06 (20), 169.01 (81), 125.02 (100)

-0.77

acid-4-O-(O-trihydroxybenzoyl)

(19), 169.01 (12), 125.02 (26)

glucopyranoside 84

7.46

C15H18O8

-

325.09268

85

7.50

C10H8O2

+

161.05942

86

7.50

C10H10O3

-

87

7.52

C9H8O4

-

88

7.63

C75H62O30

-

89

7.64

C16H20O9

90

7.65

C16H18O8

91

7.67

C90H74O36

-

92 a

7.69

C15H22O5

93 a

7.73

C15H14O6

265.07 (15), 235.06 (6), 205.05 (15), 163.04 (31),

-0.65


133.06 (56), 115.05 (17), 105.07 (24), 91.05 (4)

-1.80


177.05484

159.04 (100), 133.06 (3), 131.05 (7), 115.05 (58)

-4.97

Isosclerone isomer

179.03444

135.04 (66), 123.04 (58), 109.03 (24), 96.96 (100)

-3.02

Caffeic acid isomer

720.15863

407.07 (7), 289.07 (16), 245.08 (8), 151.04 (21),

[M-2H]2-

125.02 (100)

-0.54


-

355.10333

175.04 (56), 160.01 (89), 147.04 (12), 134.04 (100)

-0.37

Juglanoside E isomer

+

339.10727

321.10 (17), 177.05 (100), 145.03 (52), 117.03 (23)

-0.50


864.18921

425.09 (4), 407.08 (20), 289.07 (18), 245.08 (9),

[M-2H]2-

151.04 (17), 125.02 (100)

-1.74

Procyanidin hexamer

-

281.13907

237.15 (42), 189.13 (30), 171.12 (93), 123.08 (100)

-1.35

Dihydrophaseic acid

-

289.07162

245.08 (14), 221.08 (11), 203.07 (26), 151.04 (23),

-0.48

(-)-Epicatechin

145.03 (100), 119.05 (79)


Page 35 of 48


137.02 (26), 125.02 (40), 123.04 (86), 109.03 (100) 94 95

a

7.74

C27H24O18

-

635.08838

7.81

C22H18O11

-

457.07718

96

7.88

C37H30O16

-

729.14569

97

7.92

C12H6O5

+

231.02838

98

7.94

C27H24O18

-

635.08917

99

8.01

C45H38O18

-

865.19794

100

8.11

C27H24O18

-

635.08972

101

8.12

C63H44O35

-

102 a

8.12

C8H8O3

-

151.03935

103

8.14

C16H20O9

-

104

8.15

C21H22O12

-

Malabathrin A isomer

136.02 (100), 108.02 (11)

-4.77

Vanillin

355.10315

175.04 (47), 160.01 (79), 147.04 (16), 134.04 (100)

-0.87


465.10333

301.00 (8), 285.04 (53), 151.00 (100), 107.01 (59)

-1.12

425.09 (2), 407.08 (10), 289.07 (9), 245.08 (1), 151.02 (20), 125.02 (100) 313.06 (7), 271.05 (10), 211.02 (21), 169.01 (83), 125.02 (100) 301.00 (44), 289.07 (25), 275.02 (34), 229.01 (13),

[M-2H]

169.01 (47), 125.02 (100)

2-

C10H8O3

-

175.03920

107

8.19

C16H18O8

+

339.10715

-

5,8-dione isomer

-2.00

125.02 (100)

679.07452

8.19

C75H62O30

4,9-Dihydroxynaphtho[2,3-c]furan-

Trigalloyl-glucose

106

8.24

313.06 (10), 271.05 (2), 211.02 (9), 169.01 (82),

-1.82

(epi)catechin

1.15

865.19733

109

203.03 (39), 175.04 (10), 147.04 (11)

3-O-Galloyl(epi)catechin-(4,8’)-

Procyanidin trimer

-

-

169.01 (14), 151.04 (11), 125.02 (100)

-0.58

(-)-Epigallocatechin gallate

-0.69

C45H38O18

C37H30O16

-0.98 577.14 (6), 407.08 (12), 289.07 (14), 245.08 (6),

Trigalloyl-glucose

Trigalloyl-glucose

8.18

8.23

-0.96

0.28

105

108

313.06 (19), 211.02 (7), 169.01 (81), 125.02 (100)

729.14545

407.08 (10), 289.07 (10), 245.08 (2), 151.02 (17),

isomer

-1.40

Procyanidin trimer

147.04 (12), 131.05 (22)

-4.97


243.06 (13), 201.05 (13), 189.05 (18), 177.05 (100)

-0.86


125.02 (100)

407.08 (58), 289.07 (21), 245.08 (9), 169.01 (13), 151.04 (17), 125.02 (100)

720.15863

407.08 (10), 289.07 (10), 245.08 (6), 151.04 (18),

[M-2H]

125.02 (100)

2-

Quercetin-3-O-galactopyranoside


-0.91 -0.54

(Epi)catechin-(4,8’)-3’-O-galloyl(epi)catechin Procyanidin pentamer


110

8.26

C34H26O22

-

785.08698

111

8.30

C10H10O3

+

179.07022

112

8.38

C10H10O4

-

193.04993

113

8.48

C37H30O16

-

729.14575

8.53

C9H10O5

-

197.04495

114

a

301.00 (100), 275.02 (20), 257.01 (8), 229.01 (12), 169.01 (7), 125.02 (13) 161.06 (15), 149.06 (87), 143.05 (7), 133.06 (12), 117.07 (100), 115.05 (26), 105.07 (5) 175.04 (100), 147.04 (9) 407.08 (26), 289.07 (21), 245.08 (9), 169.01 (11), 151.04 (17), 125.02 (100)

551.03857

301.00 (100), 275.02 (23), 257.01 (13), 229.01

[M-2H]2-

(18), 169.01 (22), 125.02 (40)

115

8.54

C48H32O31

-

116

8.56

C27H24O18

-

635.08826

117 a

8.58

C9H8O3

-

163.03908

118

8.67

C34H26O22

-

785.08099

119

8.68

C9H8O4

-

179.03424

120

8.72

C22H18O10

-

441.08316

121

8.75

C37H30O16

-

729.14502

313.06 (16), 301.00 (4), 211.02 (5), 169.01 (80), 125.02 (100) 119.05 (100), 93.03 (3) 301.00 (100), 275.02 (21), 257.01 (12), 229.01 (22), 169.01 (7), 125.02 (11) 109.03 (96), 96.96 (100) 289.08 (63), 245.08 (35), 205.05 (25), 203.07 (47), 151.04 (41), 125.02 (74), 109.03 (100) 577.14 (5), 407.08 (21), 289.07 (15), 245.08 (9), 169.01 (11), 151.04 (15), 125.02 (100)

720.15833

407.08 (7), 289.07 (11), 245.08 (5), 151.04 (18),

[M-2H]2-

125.02 (100) 166.03 (43), 151.00 (100), 123.01 (27)

122

8.80

C75H62O30

-

123 a

8.88

C9H10O4

-

181.04997

124

8.92

C20H20O11

-

435.09280

125

8.94

C40H32O15

-

751.16602

285.04 (46), 179.00 (17), 152.01 (32), 151.00 (100), 125.02 (32), 107.01 (58) 461.08 (39), 289.07 (76), 245.08 (22), 205.05 (23), 203.07 (36), 125.02 (100), 109.03 (62)


Page 36 of 48

3.41


-0.28

Juglanside isomer

-3.63

Ferulic acid isomer

-0.49

(Epi)catechin-(4,8’)-3’-O-galloyl(epi)catechin

-3.04

Ethyl gallate

-0.96

Calamanin A isomer

-1.15

Trigalloyl-glucose

-6.07

p-Coumaric acid

-4.22


-4.13

Caffeic acid isomer

1.00

Epicatechin gallate isomer

-1.49


-0.96


-3.65

Syringaldehyde

-1.10 -1.09

Taxifolin-3-O-arabinofuranoside isomer Galloyl catechin isomer

Page 37 of 48


126

9.01

C37H30O16

-

729.14581

127

9.03

C44H34O20

-

881.15613

128

9.12

C10H10O3

-

177.05484

129

9.14

C20H20O11

-

435.09268

130

9.21

C21H22O12

-

465.10342

131

9.29

C34H28O22

-

787.09979

132 a

9.42

C10H10O4

-

193.04987

134

9.44

C44H34O20

-

135

9.44

C82H56O52

-

136

9.48

C19H14O12

137

9.50

138

881.15601

577.14 (9), 407.08 (27), 289.07 (24), 245.08 (9), 205.05 (8), 169.01 (8), 151.04 (24), 125.02 (100) 729.14 (11), 407.08 (42), 289.07 (37), 245.08 (8), 203.07 (11), 169.01 (25), 125.02 (100) 159.04 (6), 133.06 (100), 131.05 (5), 115.05 (2) 285.04 (58), 179.00 (14), 151.00 (100), 125.02 (19), 107.01 (61) 285.04 (13), 151.00 (13), 125.02 (100) 635.09 (5), 465.07 (3), 313.06 (6), 169.01 (91), 125.02 (100) 407.08 (25), 289.07 (16), 245.08 (8), 169.01 (16), 125.02 (100)

-0.41


-1.07

Di(epi)gallocatechin

-4.97

Isosclerone isomer

-1.38 -0.92

Taxifolin-3-O-arabinofuranoside isomer Quercetin-3-O-galactopyranoside isomer

-0.20

Tetragalloyl-glucose

-3.94

Ferulic acid

-1.20


-0.79

Rugosin F isomer

-0.39

Ellagic acid pentoside isomer

-1.75


-1.85

Quercetin galloylhexoside isomer

-3.15


935.07886

1059.10 (1), 935.08 (6), 917.07 (4), 767.07 (1),

[M-2H]2-

301.00 (100), 284.00 (5), 257.01 (7)

-

433.04108

301.00 (59), 299.99 (100), 257.01 (2), 229.01 (3)

C34H28O22

-

787.09857

9.55

C28H24O16

-

615.09802

139

9.57

C22H18O10

-

441.08133

140

9.72

C10H10O3

+

179.07059

161.06 (100), 133.06 (26), 115.05 (3), 105.07 (6)

1.79

Juglanside

141

9.72

C10H8O2

+

161.05936

133.06 (33), 115.05 (5), 105.07 (11), 91.05 (2)

-2.17


142

9.72

C10H10O3

-

177.05487

159.04 (68), 133.06 (45), 131.05 (34), 115.05 (35)

-4.80

Isosclerone isomer

143

9.76

C34H28O22

-

787.09992

617.08 (21), 465.07 (3), 313.06 (7), 169.01 (85),

-0.04


617.08 (13), 465.07 (11), 313.06 (16), 169.01 (98), 125.02 (100) 301.03 (33), 300.03 (100), 271.04 (86), 169.01 (29), 125.02 (27) 289.07 (78), 245.08 (40), 203.07 (53), 151.04 (36), 125.02 (100), 109.03 (98)



Page 38 of 48

125.02 (100) 144

9.79

C28H24O16

-

615.09918

145

9.81

C21H22O10

-

433.11368

146 a

9.83

C22H18O10

-

441.08075

147

9.94

C21H22O11

-

449.10846

148 a 149

a

9.99

C15H12O7

-

303.05191

301.03 (41), 300.03 (100), 271.04 (71), 169.01 (20), 125.02 (21) 271.06 (60), 177.01 (9), 151.00 (100), 119.05 (72) 289.07 (11), 245.08 (5), 203.07 (7), 169.01 (50), 151.04 (7), 137.02 (9), 125.02 (100) 287.06 (19), 151.00 (81), 135.04 (100), 107.01 (25) 285.04 (16), 217.09 (7), 175.04 (18), 151.04 (12), 125.02 (100), 109.03 (6)

0.03

Quercetin galloylhexoside isomer

-0.79

Naringenin-7-O-glucoside isomer

-4.47

(-)-Epicatechin gallate

-1.05

Astilbin isomer

2.90

Taxifolin

-0.60

Myricitrin

317.03 (22), 316.02 (100), 287.02 (51), 271.02 10.00

C21H20O12

-

463.08792

(85), 259.02 (17), 179.00 (11), 151.00 (18), 125.02 (4), 109.03 (4)

150

10.06

C37H30O16

-

729.14532

151 a

10.14

C21H20O12

-

463.08817

152

a

577.14 (6), 407.08 (13), 289.07 (10), 245.08 (6), 169.01 (13), 151.04 (10), 125.02 (100) 301.03 (24), 300.03 (6), 255.03 (38), 243.03 (22), 227.03 (7), 179.00 (5), 151.00 (16)

-1.08


-0.06

Hyperoside

-0.90

Ellagic acid

284.00 (17), 271.02 (100), 257.01 (76), 255.03 10.17

C14H6O8

-

300.99872

(40), 243.03 (24), 229.01 (13), 179.00 (5), 151.00

153

10.33

C15H12O7

-

303.05084

285.04 (15), 217.05 (7), 175.04 (20), 125.02 (100)

-0.63

Taxifolin isomer

154

10.34

C23H22O12

-

489.10336

327.05 (18), 229.05 (2), 175.04 (100)

-1.00

3’-O-acetylquercitrin isomer

155 a

10.35

C21H20O12

-

463.08719

-2.18

Isoquercitrin

156 a

10.36

C21H20O11

-

447.09305

-0.51

Luteoloside

157 a

10.40

C21H22O11

-

449.10840

-1.18

Astilbin

158

10.40

C28H24O15

-

599.10657

3.89

Quercetin galloyl deoxyhexoside

(16)

301.03 (25), 300.03 (75), 271.02 (100), 227.03 (6), 179.00 (4), 151.00 (15) 303.05 (8), 285.04 (43), 179.00 (19), 151.00 (100), 137.02 (25), 125.02 (39), 107.01 (57) 325.03 (16), 285.04 (20), 255.03 (29), 227.03 (17),


Page 39 of 48


169.01 (68), 125.02 (100) 159 a

10.43

C20H20O11

-

435.09125

160

10.48

C22H22O12

-

477.10382

161

10.55

C22H18O10

-

441.08295

162

10.70

C20H18O11

-

433.07724

163

10.72

C10H10O3

-

177.05496

164

10.73

C10H10O3

+

179.06996

165

10.75

C10H6O4

-

189.01854

166

10.90

C20H18O11

-

167

10.92

C13H16O10

168

10.94

C11H12O4

303.05 (11), 285.04 (46), 179.00 (24), 151.00 (100),

-4.67

Taxifolin-3-O-arabinofuranoside

-0.06

Isorhamnetin-3-O-glucoside isomer

0.52


301.01 (3), 300.03 (5), 271.03 (8)

-0.90

Quercetin pentoside isomer

159.04 (3), 133.06 (4), 115.05 (5)

-4.29

Isosclerone isomer

-1.73

Juglanside isomer

161.02 (100), 117.03 (11)

-4.18

Dihydroxy-naphthoquinone isomer

433.07748

301.01 (17), 300.03 (85), 271.02 (100)

-0.35


-

331.06693

271.04 (46), 211.02 (100), 169.01 (69), 125.02 (94)

-0.42

Monogalloyl-glucose

-

207.06543

192.04 (29), 177.02 (100), 149.02 (33), 123.04(13)

-4.10

Sinapinaldehyde

-0.40

Quercetin galloyl deoxyhexoside

0.03

Monogalloyl-glucose

-0.61


169

11.07

C28H24O15

-

599.10400

170

11.08

C13H16O10

-

331.06708

171

11.08

C23H22O12

-

489.10355

172

11.09

C23H22O12

+

491.11810

173

11.19

C20H18O11

-

433.07709

174

11.23

C23H22O12

-

489.10333

175

11.30

C21H22O10

-

433.11356

125.02 (53), 107.01 (57) 265.07 (15), 205.05 (28), 177.06 (12), 163.04 (56), 145.03 (80), 125.02 (100), 119.05 (83) 289.07 (56), 245.08 (40), 205.05 (26), 203.07 (36), 151.04 (42), 125.02 (98), 109.03 (100)

161.06 (60), 147.04 (100), 133.06 (23), 119.05 (53), 105.07 (18)

313.06 (9), 285.04 (26), 284.03(34), 227.03 (24), 169.01 (78), 125.02 (100) 271.05 (44), 211.02 (100), 169.01 (88), 125.02 (96) 313.06 (14), 271.05 (17), 229.05 (6), 175.04 (100), 169.01 (68), 125.02 (69) 297.06 (3), 201.05 (3), 153.02 (100) 300.03 (3), 271.06 (60), 169.01 (5), 151.00 (69), 119.05 (100) 313.06 (14), 271.05 (6), 175.04 (100), 169.01 (65), 125.02 (84) 271.06 (60), 177.01 (9), 151.00 (69), 119.05 (100)


-0.61

Trihydroxynaphthalene-O(O-trihydroxybenzoyl) glucoside

-1.25


-1.06


-1.06

Naringenin-7-O-glucoside isomer


Page 40 of 48

301.04 (43), 300.03 (78), 271.02 (100), 255.03 176

a

11.41

C21H20O11

-

447.09305

(52), 243.03 (20), 227.03 (10), 179.00 (9), 151.00

-0.51

Quercitrin

(26) 177

11.45

C37H30O16

-

729.14484

178

11.46

C9H16O4

-

187.09737

179

11.56

C13H22O2

+

211.16989

180

11.60

C22H22O12

-

477.10406

181

11.80

C21H22O11

-

449.10861

182

11.81

C10H10O4

-

193.04979

183

12.02

C44H34O20

-

881.15594

184

12.15

C28H24O14

-

583.10962

185 a

12.17

C21H24O10

-

435.12930

186

12.26

C10H6O4

-

189.01862

187

12.27

C23H20O12

+

489.10138

188

12.63

C21H20O10

-

431.09775

189

13.00

C22H22O12

-

477.10300

190

13.85

C25H26O12

-

517.13434

407.08 (38), 289.07 (21), 245.08 (9), 203.07 (11), 169.01 (13), 151.04 (17), 125.02 (100) 169.09 (3), 125.10 (100), 97.06 (23) 193.16 (21), 175.15 (25), 135.12 (76), 119.09 (26), 109.10 (100) 314.04 (12), 285.04 (9), 271.02 (20), 257.05 (7), 243.03 (24), 169.01 (52), 125.02 (100) 301.03 (4), 287.06 (17), 151.00 (83), 135.04 (100), 107.01 (29) 178.03 (100), 123.01 (12), 108.02 (18) 729.15 (7), 407.08 (23), 289.07 (14), 245.08 (5), 169.01 (14), 125.02 (100) 301.03 (29), 300.03 (84), 271.02 (100), 255.03 (43), 151.00 (16) 273.08 (35), 179.03 (10), 167.03 (100), 151.00 (7),

-1.74 -1.12 2.98

(Epi)catechin-(4,8’)-3’-O-galloyl(epi)catechin Azelaic acid 9-Hydroxymegastigman-4-en-3-one isomer

0.44

Isorhamnetin-3-O-glucoside isomer

-0.71

Astilbin isomer

-4.35

Ferulic acid isomer

-1.28


0.50

Quercetin-O-(p-hydroxy)benzoylhexoside

-0.85

Phlorizin

161.02 (100), 117.03 (19)

-3.76

Dihydroxy-naphthoquinone isomer

327.04 (100), 309.04 (8), 265.05 (24), 237.05 (23)

-2.80

Jugnaphthalenoside A

-1.44

Afzelin

315.05 (27), 300.03 (100), 271.02 (75), 255.03 (31)

-1.78

Isorhamnetin-3-O-glucoside isomer Dihydroxynaphthol-O-[O-

175.04 (100), 131.05 (11)

-1.57

(dimethoxy-hydroxybenzoyl)]

125.02 (26), 123.04 (71), 119.05 (20), 93.03 (15)

285.04 (41), 284.03 (41), 257.05 (5), 255.03 (100), 229.05 (14), 227.03 (70)

glucopyranoside


Page 41 of 48


191

13.86

C13H22O2

+

211.16989

a

14.05

C15H10O7

-

301.03564

193 a

14.10

C15H10O6

-

285.04095

194 a

15.06

C15H12O5

-

271.06110

195

15.18

C20H22O5

-

341.13937

192

196 a

15.29

C15H14O5

-

273.07675

197

15.70

C16H12O6

-

299.05545

198

17.60

C20H22O5

-

341.13953

199 a

17.94

C15H10O4

-

253.05043

a

18.45

C16H12O6

-

299.05579

200 a

193.16 (22), 175.15 (21), 135.12 (65), 119.09 (24), 109.10 (100) 300.00 (11), 179.00 (23), 151.00 (100), 107.01 (58) 217.05 (5), 175.04 (12), 151.00 (21), 133.03 (100), 107.01 (18) 187.04 (4), 177.02 (6), 165.02 (2), 151.00 (44), 119.05 (100), 107.01 (34), 93.03 (13) 326.12 (100), 309.11 (10), 253.05 (16), 225.06 (35) 189.05 (11), 179.03 (4), 167.03 (90), 151.00 (11), 125.02 (25), 123.04 (90), 119.05 (70), 93.03 (14) 284.03 (100), 256.04 (48), 227.03 (10), 151.00 (6) 326.12 (69), 219.07 (38), 177.06 (83), 151.04 (21), 137.02 (66), 135.04 (52), 121.03 (100) 209.06 (9), 167.05 (5), 143.05 (21), 107.01 (9)

Compared with reference standards


2.98

9-Hydroxymegastigman-4-en-3-one isomer

0.86

Quercetin

1.72

Luteolin

-0.37

Naringenin

-0.23

Caffeoyl glucopyranose isomer

-0.37

Phloretin

-2.21

Kaempferide isomer

0.23

Caffeoyl glucopyranose isomer

-0.79

Chrysin

-1.07

Kaempferide


Page 42 of 48

Table 2. Contents of 21 dietary polyphenols in ten batches of Diaphragma juglandis fructus (μg/g of dry sample) Compounds

XJ-1

XJ-2

XJ-3

SD-1

SD-2

SD-3

HB-1

HB-2

SX-1

SX-2

Gallic acid

149.90

216.50

139.06

219.09

89.87

165.46

191.38

160.31

205.27

184.78

Protocatehuic acid

130.64

154.04

122.89

146.89

99.14

44.28

105.43

97.14

119.14

115.24

Neochlorogenic acid

32.49

56.04

69.97

99.68

34.00

84.96

126.57

66.90

93.81

108.15

Protocatechualdehyde

34.09

30.11

29.69

34.60

24.95

9.79

22.80

25.97

24.52

24.06

Methyl gallate

75.71

216.29

181.34

151.65

66.77

34.32

63.83

155.20

355.66

260.26

(+)-Catechin

271.14

336.24

365.02

579.34

289.19

693.32

251.69

404.11

429.55

577.42

Vanillic acid

20.27

27.40

23.45

25.33

18.68

34.59

44.13

24.15

21.32

20.77

Syringic acid

13.24

21.96

13.90

16.83

11.44

26.76

13.48

17.29

14.74

16.51

(-)-Epicatechin (-)-Epigallocatechin gallate (-)-Epicatechin gallate

16.72

9.50

24.51

33.37

14.11

36.44

8.82

18.98

30.86

32.09

0.50

0.43

0.72

1.11

0.59

1.06

2.21

0.78

0.93

1.66

99.66

63.47

111.87

143.37

82.97

146.05

22.30

127.95

176.79

194.79

Taxifolin

33.99

51.71

52.12

114.75

41.28

87.77

153.31

61.78

93.78

121.32

Myricitrin

11.22

12.65

19.12

22.37

11.86

19.27

28.13

24.24

14.51

23.83

Hyperoside

31.80

26.30

60.27

19.47

31.04

35.47

4.20

35.12

18.25

20.03

Ellagic acid

1412.43

1733.64

1419.01

1332.03

1001.97

518.38

742.61

1003.97

1453.22

1466.24

Isoquercitrin

45.20

116.68

64.12

62.92

32.72

32.38

20.89

52.18

47.23

62.84

Astilbin

9.19

14.48

14.38

26.17

9.90

16.97

23.88

15.13

27.33

31.62

Taxifolin-3-O-arabinofuranoside

544.39

985.73

844.92

1884.65

519.60

784.59

1549.24

920.83

1758.58

2181.84

Quercitrin

544.88

801.09

795.26

634.93

478.06

664.26

145.42

983.58

603.99

691.41

Phlorizin

8.54

20.87

10.07

34.36

8.35

3.18

21.92

17.83

29.81

34.57

Quercetin

12.18

14.78

16.90

11.02

11.65

14.58

4.76

17.43

13.06

14.07

Total

3498.18

4909.91

4378.59

5593.93

2878.14

3453.88

3547.00

4230.87

5532.35

6183.50


Page 43 of 48


Figure 1



Figure 2


Page 44 of 48

Intensit

1.6E+9 1

34

0

2

4

198 199

190

159

56 46

194

151,152

33 39,40

10 68 7 11

Figure 3 0.0E+0

79 38 197 145,146 192 37 70 of Agricultural and Food Chemistry 27 Journal

14

2 5 9 13

8.0E+8

200 193

161

60

6

8

10

12

14

16

18

6.0E+9

90

Intensity (cps)

3.6E+9

31

140,141

45

164

74

191

12

0

2

4

6

23 22 21 24-26

4.0E+9

20 19

3.2E+9

18 Intensity (cps)

172

187

17

1.2E+9

16

2.4E+9

4 3

1.6E+9 1

0

8

10 Time (min)

15

132-137 131

79 38 27 37 70 34

14

2 5 9 13

16

46

18

197 198 199

190

200 193

161

60

6

8

10 Time (min)

12

14

16

18

6.0E+9

20

22

Positive

90

4.8E+9

85 3.6E+9

81

111

97

179 172

107 140,141

187

17

2.4E+9

31

45

164

191

74

12

1.2E+9

0.0E+0

22

195 196

194

159

20

Negative

192

151,152

56

4

14

189

145,146

33 39,40

2

12

181,182 98-112 180 184,185 177,178 113 183 106 114 186 95,96 121,122 188 126,127 91-94 88,89 128 86,87

10 8 6 7 11

8.0E+8

0.0E+0

22

179

107

81

2.4E+9

111

97

85

0.0E+0

20

Positive

4.8E+9

Intensity (cps)

Page 45 of 48

3

0

2

4

6

8

10 Time (min)

12

14


16

18

20

22


Page 46 of 48

Figure 4

(A) Relative Abundance

100

DPI 300.99887

Compound 3 [M-H] 481.06226

50 229.01390

-glucose 0

100

200

300 m/z

400

500

600

(B) Relative Abundance

100

DPI 125.02339 DPI 169.01329

50

Compound 2 [M-H] 331.06766 -glucose

0

100

200

m/z


300

400

Page 47 of 48


Figure 5 5

7x10

5

6x10

5

5

4x10

5

3x10

Intensity (cps)

5x10

5

2x10

5

1x10 0

Co

mp

ou

nd

NO .

C8 C24 C20 C42 C33 C61 C56 C93 C76 C146 C95 C149 C148 C152 C151 C157 C155 C176 C159 C192 C185 0

1

2

3

4

5

6

7

8

9

10 11 12 Time (min)

13

14

15

16

17

18


19

20


Table of Contents Graphic (TOC)


Page 48 of 48

Identification and Quantification of Bioactive Compounds in

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