Comparison of Chemical Profiling and Antioxidant Activities of Fruits

Oct 22, 2014 - ABSTRACT: Citrus grandis 'Tomentosa' (CGT) is particularly cultivated in China and widely used in health foods. In this study, the chem...
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Comparison of chemical profiling and antioxidant activities of fruits, leaves, branches and flowers of Citrus grandis ‘Tomentosa’ Li Duan, Long Guo, Li-Li Dou, Ke-Yun Yu, E-Hu Liu, and Ping Li J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/jf5036355 • Publication Date (Web): 22 Oct 2014 Downloaded from http://pubs.acs.org on November 8, 2014

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Journal of Agricultural and Food Chemistry

Comparison of chemical profiling and antioxidant activities of fruits, leaves, branches and flowers of Citrus grandis ‘Tomentosa’

Li Duan, Long Guo, Li-Li Dou, Ke-Yun Yu, E-Hu Liu*, Ping Li*

State Key Laboratory of Natural Medicines, China Pharmaceutical University, Nanjing, P. R. China

AUTHOR INFORMATION Corresponding Authors *E-Hu Liu, E-mail: [email protected]. Phone: +86 25 83271379. *Ping Li, E-mail: [email protected]. Phone: +86 25 83271379.

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ABSTRACT

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Citrus grandis ‘Tomentosa’ (CGT) is particularly cultivated in China and widely used

3

in health foods. In this study, the chemical profiles of different parts of CGT were

4

comprehensively compared by rapid resolution liquid chromatography coupled to

5

electrospray ionization quadrupole time-of-flight mass spectrometry method. A total

6

of 22 compounds were identified and two C-glucosyl flavones were found for the first

7

time in CGT. Four main constituents (rhiofolin, naringin, meranzin hydrate and

8

isoimperatorin) in different parts of CGT were simultaneously determined. Overall,

9

the contents of the four main compounds decreased with the ripening process. In

10

parallel, the antioxidant activities of their extracts were also evaluated by three assays

11

(ABTS, DPPH, FRAP), and the results indicated a similar tendency: small fruit >

12

flower ~ medium fruit > large fruit > leaf ~ branch. The results obtained in the present

13

work may provide useful information for future utilization of CGT.

14

Citrus

grandis

‘Tomentosa’;

15

KEYWORDS:

16

RRLC-ESI-QTOF-MS/MS; Antioxidant activity; Flavanoids

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Chemical

profiling;

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INTRODUCTION

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The Citrus genus is one of the most widely cultivated crops in the world because

20

of its good taste and nutritional benefits.1 Fresh citrus fruit and citrus-derived products

21

have been the essential ingredients of human diet. Citrus grandis (L.) Osbeck, one of

22

three original citrus species, has been planted more than 200 cultivars in southern

23

China.2 C. grandis ‘Tomentosa’ (abbreviated as CGT) is a cultivar of C. grandis (L.)

24

Osbeck, particularly originated from Huazhou town in Guangdong province, China.3

25

CGT has been of particular interest due to its important pharmacological effects,

26

including antioxidant, anti-inflammatory, antitussive and expectorant activities.4-6

27

Phytochemical studies demonstrated that the fruits of CGT possess high amounts of

28

bioactive compounds which can influence human health, e.g. carbohydrates, minerals,

29

phenolic acid, flavonoids and limonoids.7-10

30

Several studies have been carried out to characterize the chemical components in

31

fruits of CGT, revealing some main constituents, such as flavanone O-glycosides,

32

flavone C- and O-glycosides, coumarins and limonoids.11,12 However, little is known

33

about whether the harvest time, resulting in different size of fruits, can influence the

34

composition and antioxidant activities of CGT. Also, most of researches mainly focus

35

on the fruits, but the chemical compounds in the different parts (leaves, branches and

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flowers) of CGT have never been studied.

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In the past few years, high performance liquid chromatography and liquid

38

chromatography coupled to mass spectrometry have been widely accepted to be the

39

predominant tool for target and non-target analysis of chemical constituents in plant

40

materials.13,14 Hence, a rapid resolution liquid chromatography coupled to

41

electrospray

ionization

quadrupole

time-of-flight 3

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(RRLC-ESI-QTOF/MS) method was developed to comprehensively compare the

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chemical constituents of different parts of CGT. The contents of main constituents in

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CGT were quantified by RRLC combined with segmental monitoring strategy, and

45

the antioxidant activities of different parts were also evaluated by three in vitro

46

assays.

47 48

MATERIALS AND METHODS

49 50

Plant Materials.

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Citrus grandis ‘Tomentosa’ plant materials (fruits, flowers, leaves and branches),

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obtained from Huazhou city in 2013, were kindly supplied by Huazhou Pummelo Peel

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Medical Materials Development Co., Ltd., China. Fruits were harvested from April to

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June. Based on the size and harvest time, fruits were sampled randomly and divided

55

into three groups: Small (diameter 1-3 cm, samples labeled S1-8), Medium (diameter

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4-5 cm, samples labeled M1-6) and Large (diameter 6-8 cm, samples labeled L1-6).

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Flowers were collected in February, while leaves and branches were freshly picked in

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April. The fruits, flowers, leaves and branches were dried under sunlight and then

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ground into fine powder.

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Chemicals and Reagents.

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Reference compounds of naringin, rhoifolin, meranzin hydrate, isoimperatorin

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and gallic acid were purchased from Chengdu Must Bio-technology Co., Ltd.

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(Chengdu, China). Total antioxidant capacity assay kits (ABTS method and FRAP

65

method) were purchased by Beyotime Institute of Biotechnology (S0121 and S0116,

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Shanghai, China). 2,2-diphenyl-1-picrylhydrazyl (DPPH) and Folin–Ciocalteu reagent 4

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were purchased from Sigma-Aldrich (St. Louis, MO, USA). Acetonitrile of HPLC

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grade was purchased from ROE (Newark, New Castle, USA). HPLC-grade methanol

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was obtained from Burdick & Jackson Honeywell International Inc. (Muskegon, MI,

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USA). Deionized water used in experiments was purified by a Milli-Q system

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(Millipore, Milford, MA, USA). All other reagents and chemicals used were of

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analytical grade.

73 74

Sample Preparation and Standard Solution.

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Each sample (fruits, flowers, leaves and branches) powder (0.5 g) was weighted

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accurately and extracted by ultrasonator with 25 mL methanol for 30 min at 100 Hz.

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The supernants were centrifuged at room temperature for 10 min at 16200 G, and

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different samples were diluted different times (6 times for small fruits and flowers, 3

79

times for medium and large fruits) with methanol before use. An aliquot of 2 µL of

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the filtrate was injected for RRLC analysis and 1 µL for MS analysis.

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Standard stock solutions of naringin, rhoifolin, meranzin hydrate and

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isoimperatorin were prepared at concentrations of 1000, 800, 200 and 200 µg/mL,

83

respectively, and stored at -20 °C. Prior to injection, stock solutions were

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appropriately diluted with methanol to a series of appropriate concentrations used as

85

working solutions.

86 87

RRLC-ESI-MS/MS Analysis.

88

Analysis was performed on an Agilent 1260 series HPLC system coupled with a

89

6530 Accurate-Mass Q-TOF system with a Dual AJS ESI source (Agilent

90

Technologies, Palo Alto, CA, USA). Samples were separated on an Agilent ZORBAX

91

SB-C18 column (4.6×50 mm, 1.8 µm) at 25 °C. Mobile phase consisted of 0.1% 5

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aqueous formic acid (A) and acetonitrile (B) using a gradient program of 25% (B) in

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0-3 min, 25-90% (B) in 3-14 min, 90-100% (B) in 14-15 min. The flow rate was 0.5

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mL/min.

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The conditions of ion source were as follows: nebulizer gas setting at 45 psig,

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capillary voltage at 3000 V, fragmentor voltage at 120 V, drying gas (N2) flow rate

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and temperature at 10 L/min and 325 °C, sheath gas flow rate and temperature at 11

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L/min and 350 °C. MS acquisition was performed by using electrospray inonization

99

(ESI) in positive ion mode. Collision energy was set at 20 V. All the data were

100

acquired using the Extended Dynamic Range mode (2 GHz) and the mass range was

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set at 100-1000 Da in centroid mode. The TOF was calibrated every day before

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sample analysis and, subsequently, used reference masses at m/z 121.0508 and

103

922.0098 to obtain high-accuracy mass measurements.

104 105

RRLC-DAD Analysis.

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Instrument and chromatographic conditions.

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Quantitative analysis was performed on an Agilent 1260 Infinity LC system

108

equipped with a photo diode array detector. Samples were separated on an Agilent

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ZORBAX SB-C18 column (4.6×50 mm, 1.8 µm) at 25 °C. Mobile phase consisted of

110

0.1% aqueous formic acid (A) and methanol (B) using a gradient program of 35-39%

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(B) in 0-3 min, 39-39% (B) in 3-5 min, 39-55% (B) in 5-12 min, 55-90% (B) in 12-21

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min, 90-100% (B) in 21-23min. The flow rate was 0.5 mL/min. Different detection

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wavelengths were performed for different periods of time: 283 nm for 0-9 min, 330

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nm for 9-18 min and 310 nm for 18-23 min.

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Method validation.

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An aliquot of 2 µL of working solutions were injected into RRLC for analysis, 6

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the calibration curves were constructed by plotting the peak areas versus the

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concentration of each analyte. The limit of detection (LOD) and limit of

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quantification (LOQ) for each analyte were defined by the concentrations that

120

generated peaks with signal-to-noise values (S/N) of 3 and 10, respectively.

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The precision of the developed method was determined by the intra- and

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inter-day variations. For intra-day test, the samples were analysed for six times within

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the same day, while for inter-day test, the samples were examined in duplicates for

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consecutive three days.

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To confirm the repeatability, six replicates of the same samples were extracted

126

and analyzed. For the stability test, the same sample was stored at room temperature

127

and analyzed by replicate injection at 0, 2, 4, 8, 16 and 24 h. The RSDs were used to

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evaluate the method repeatability and stability. Accuracy was calculated as the

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percentage of recovery. Recovery tests were performed by spiking a known amount of

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the four standards to 0.25 g powder and then extracted and analyzed with the same

131

procedures. Six replicates were performed for the test.

132 133

Determination of Total Phenolics.

134

Total phenolic contents (TPC) of each sample were determined by using the

135

Folin–Ciocalteu reagent according to Singleton et al. with slight modifications.15,16

136

Gallic acid was used to prepare a standard curve, and final values were expressed in

137

milligramme gallic acid equivalents (mg GAE/g). Diluted CGT extracts were

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incubated with Folin−Ciocalteu phenol reagent. The color was developed by adding

139

sodium carbonate (10%) and absorbance was measured at 765 nm.

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Antioxidant Assays. 7

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Free Radical Scavenging Capacity.

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The free radical scavenging capacity was analyzed by the DPPH assay.17,18 DPPH

144

is a stable organic nitrogen radical with purple color. Upon reduction by an

145

antioxidant, the solution color faded and the reaction can be monitored by a

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spectrophotometer at 517 nm. The decrease in absorbance was proportional to the

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antioxidant capacity and can be measured in comparison to Trolox as standard. All

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samples and standard were diluted in methanol. 100 µL of CGT extracts were mixed

149

with 100 µL of DPPH (0.2 mM) in methanol, and the change in optical density (517

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nm) was monitored after 30 min using a microplate reader. Results were expressed as

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µM Trolox equivalents (TE) / g

152

Ferric Reducing Antioxidant Power Assay.

153

The FRAP assay was carried out by a commercial kit (Beyotime Inc., China)

154

following the instruction manual.19,20 Under acidic conditions, antioxidants can

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restore ferric-tripyridyltriazine (Fe3+-TPTZ) into blue Fe2+-TPTZ. The total

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antioxidant activity of antioxidants can be reflected by the absorbance of Fe2+-TPTZ

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at 593 nm. Absorbance was measured at 593 nm using a microplate reader, with

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FeSO4 as the antioxidant standard. The results of the FRAP assay were calculated as

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mM FeSO4 / g powder.

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ABTS Radical Scavenging Activity Assay.

161

The ABTS assay was performed by a commercial kit (Beyotime Inc., China)

162

following the instruction manual.21,22 With the appropriate oxidants, ABTS can be

163

oxidized into green ABTS·+, while antioxidants can inhibit this process. The total

164

antioxidant activity of antioxidants can be reflected by the absorbance of ABTS·+ at

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734 nm. Absorbance was measured at 734 nm using a microplate reader, with Trolox

166

as an antioxidant standard. The results were calculated as µM Trolox Equivalent (TE) 8

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/ g powder.

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Statistical Analysis.

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All determinations of antioxidant capacity were conducted in triplicates. The values

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expressed were the mean of three measurements. Statistical comparisons of the results

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were performed by one-way ANOVA using SPSS 11.5 (Chicago, IL). Significant

173

differences (p < 0.05) between the different kinds of samples were analyzed by

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Duncan’s multiple range test.

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RESULTS AND DISCUSSION

177 178 179

Comparison of Chemical Constituents of Different Parts of CGT. The

profiling

of

CGT

chemical

constituents

was

analyzed

by

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RRLC-ESI-QTOF/MS method (Figure 1). Based on their retention time and MS

181

fragmentation behavior, a total of 22 compounds, including flavanone O-glycosides

182

(peaks 6, 7, 9, 10), flavanone aglycone (peaks 12), flavone C- (peaks 1, 2, 3, 5) and

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O-glycosides (peaks 4, 8), coumarins (peaks 11, 13-16, 21, 22) and limonoids

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(peaks17-20) were identified or tentatively characterized (Table 1).

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Flavonoid glycosides are divided into two categories: flavonoid O- and

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C-glycosides. Favonoid-O-glycosides ruptures are easily induced by loss of the sugar

187

moiety, while fragmentations in C-glycosides focus preferentially on the glycidic

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moiety.23,24 At low collision energies, the main fragments of flavonoid-C-glycosides

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were due to the loss of water ([M+H–nH2O]+), and loss of the glucosidic methylol

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group as formaldehyde ([M+H–CH2O–2H2O]+).25 In this work, ten compounds were

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elucidated as flavonoid glycosides in CGT. Noteworthyly, compound 2 and 3 were 9

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identified in CGT for the first time. Figure 2 shows the MS/MS spectra of compound

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2 and 3 in positive ion mode with 120V fragmentor voltage and 20V collision energy.

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Compound 3 yielded a [M+H]+ at m/z 579.1704 and a predominant fragment ion at

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m/z 433, corresponding to the loss of a terminal rhamnose from the protonated

196

molecule. The product ion at m/z 433 further undertook an inner sugar cleavage to

197

yield

198

([M+H-145-2H2O]+), 379 ([M+H-145-3H2O]+), and 367([M+H-145-2H2O-CH2O]+).

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The above typical fragments were in agreement with 8-C-glucosyl apigenin (vitexin)

200

or 6-C-glucosyl apigenin (isovitexin).25 Therefore, compound 3 was proposed to be

201

O-pentosyl-vitexin or -isovitexin. According to the literature data, the possible

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chemical structure of compound 3 was apigenin-8-C-glucoside-O-rhamnoside or

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apigenin-6-C-glucoside-O-rhamnoside, which has been detected in Citrus species.26-28

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Compound 2 exhibited a [M+H]+ at m/z 565.1504, showing similar MS/MS

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fragmentation pattern with compound 3. The same ion at m/z 433 due to the loss of a

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terminal arabinose from precursor ion was also observed. Compared with compound 3,

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compound 2 was plausibly identified as apigenin-8-C-glucoside-O-arabinoside or

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apigenin-6-C-glucoside-O-arabinoside.

the

following

fragments

at

m/z

415

([M+H-145-H2O]+),

397

209

Coumarin compounds are generally distributed throughout the Citrus species.10 In

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the present work, a total of seven coumarins were detected and elucidated in CGT.

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Most coumarins exhibited quasi-molecular ions [M+H]+, [M+Na]+, and fragment ions

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[M−CO+H]+, [M−2CO+H]+, [M−CO2+H]+ and [M−CO2−CO+H]+.

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Citrus limonoids have attracted much more attention in recent years because of

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their health benefits.29,30 By comparing their quasi-molecular weights, fragment ions

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and chromatographic properties with the reported compounds in citrus fruits, we

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tentatively elucidated four limonoids in CGT, namely limonin, nomilin, isoobacunoic 10

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acid and obacunone.31,32

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It has been widely reported that chemical constituents of Citrus species were

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dominated by flavonoids and limonoids.31 However, our results showed that CGT

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contained various courmarins, such as meranzin hydrate, bergaptan, imperatorin and

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isoimperatorin. We also observed that chemical constituents were more abundant in

222

fruits and flowers than in leaves and branches. Different sizes of fruits had almost the

223

same chemical constituents profile and yet varied in contents (Table 1), indicating that

224

the harvesting time had no significant effect on the composition of chemical

225

constituents. Isoobacunoic acid was only detected in fruits, but not in other parts of

226

CGT. The compound obacunone was not detected in leaves, but we found abundant

227

obacunone in fruits and flowers.

228

In summary, the composition and content of chemical constituents varied in

229

different parts of CGT. The most abundant constituents in CGT, calculated by peak

230

area in MS spectrum, were rhiofolin, naringin, meranzin hydrate and isoimperatorin.

231 232

Quantitative Evaluation of Chemical Constituents Contents.

233

In order to develop a feasible and rapid method for quantitative analysis, different

234

mobile phase systems (ACN-water and methanol-water) were examined and

235

compared. It was showed that a good separation can be obtained in methanol/water

236

system, however, rhiofolin and naringin were not satisfactorily separated (resolution
flower ~ medium fruit > large fruit > leaf ~ branch. Phenolic compounds

301

were usually considered as the basis of antioxidant activity due to their hydroxyl

302

group.33 Similarly, our results revealed a positive relationship between contents of

303

total phenolic and antioxidant activity. In market, only fruits of CGT were consumed

304

as food and health supplement. However, our study showed that other parts of CGT,

305

especially flowers, might be used as an alternative dietary supplement.

306 307

In conclusion, we compared the chemical constituents profiles of different parts

308

of CGT by RRLC-ESI-QTOF/MS and 22 compounds were identified or tentatively

309

characterized. Of these, two C-glucosyl flavones (compound 2 and 3) were found, to

310

our best knowledge, for the first time in CGT. This may give us some new insights

311

into CGT, especially leaves and branches which have not been studied before. The

312

quantification of four major compounds by RRLC combined with segmental

313

monitoring strategy demonstrated that the contents of these compounds decreased

314

with ripening of fruits. Fruits and flowers were found to be rich in phenolic

315

compounds and had strong antioxidant activities. In particular, the discarded parts of

316

CGT, leaves and branches, also contained some bioactive compounds and might be 14

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used as an alternative dietary supplement. We hope the results may provide useful

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information for future utilization of CGT.

319 320

ABBREVATIONS USED

321

CGT, Citrus grandis ‘Tomentosa’; Q-TOF, Quadrupole time-of-flight; MS, mass

322

spectrometry; RRLC, Rapid resolution liquid chromatography; ESI, electrospray

323

inonization;

324

(3-ethylbenzthiazolinesulfonic acid) diammonium salt; FRAP, ferric reducing

325

antioxidant power; TPTZ, ferric-tripyridyltriazine; TPC, Total phenolic contents;

326

GAE, gallic acid equivalents; ANOVA, Analysis of Variance; DAD, diode array

327

detector; RSD, relative standard; TE, Trolox equivalent.

DPPH,

2,2-diphenyl-1-picrylhydrazyl;

ABTS,

2,2′-azinobis

328 329

Supporting Information Available:

330

RRLC method validation results (Tables S1 and S2). This material is available free of

331

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

332

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449 450

Funding

451

This work was supported by National Natural Science Foundation of China

452

(81473343), New Century Excellent Talents in University (NCET-13-1034),

453

Fundamental Research Funds for the Central Universities (ZD2014YW0033), Talent

454

Work Leading Group of Jiangsu Province (333 High-level Talents Training Project,

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BRA2012128), Priority Academic Program Development of Jiangsu Higher

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Education Institutions and "Six Talent Peaks Program" of Jiangsu Province of China

457

(2013-YY-001).

458

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Figure captions Figure 1. The total ion chromatograms (TIC) of different parts (fruit, flower, leaf and branch) of CGT by RRLC-MS in positive ion mode. Detection was set in positive ion mode with a 120V fragmentor. Peak numbers of compounds are in accordance with those in Table 1.

Figure 2. The MS/MS spectra of (A) compound 2 and (B) compound 3 in positive ion mode with 20V collision energy.

Figure 3. Chemical structures of the four quantitative analytes.

Figure 4. The representative RRLC chromatograms of different parts of CGT. Peak numbers of compounds are in accordance with those in Table 1.

Figure 5. The (a) total phenolic contents (TPC) and (b) ABTS, (c) FRAP and (d) DPPH, antioxidant activity of fruits, flowers, leaves and branches of CGT. Values of fruits and flowers represent means ± SD (small fruits (S): n = 8; medium fruits (M): n = 6; large fruits (L): n = 6; flower: n = 3), while values of leaves and branches represent the antioxidant activity of the only one batch we collected.

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Tables Table 1. Characterization of Chemical Constituents in Different Parts of CGT by LC-QTOF-MS/MS Analysis in Positive ion Mode. No

tR (min)

[M+H]+

Diff (ppm)

Formula

λmax (nm)

Large fruit

Medium fruit

Small fruit

Leaf

Branch

Flower

Class

1

1.416

595.1656

0.19

C27H30O15

270

++**

++

++

++

+

++

a***

++

++

++

++

+

+

a

++

++

++

++

+

+

a

255

Vicenin-2 Apigenin-8-C-glucoside-Oarabinoside or Apigenin-6-C-glucoside-Oarabinoside Apigenin-8-C-glucoside-Orhamnoside or Apigenin-6-C-glucoside-Orhamnoside Luteolin-7-O-rutinoside

2

1.927

565.1555

-0.53

C26H28O14

/#

3

2.020

579.1712

-0.64

C27H30O14

/

4

2.401

595.1668

-2.74

C27H30O15

+

+

+

++

++

+

b

5

2.430

433.1139

-2.64

C21H20O10

270

Apigenin-8-C-glucoside

++

++

++

+

+

+

a

6 7

2.627 3.238

597.1825 581.1855

-2.69 2.24

C27H32O15 C27H32O14

283 283

Eriocitrin Narirutin*

+ +

+ ++

+ ++

+

+ +

++ +++

c c

8 9

3.576 3.778

579.1716 581.1876

-0.91 -1.74

C27H30O14 C27H32O14

330 283

Rhiofolin* Naringin*

+++ +++

+++ +++

+++ +++

+++ ++

++ ++

+++ +++

b c

10

5.733

725.2273

1.62

C33H40O18

280

Melitidin

+

+

+

+

+

+

c

Identification

*

11

6.208

279.1228

-0.49

C15H18O5

330

Meranzin hydrate

+++

+++

+++

+

+

+

d

12

9.111

273.0756

0.39

C15H12O5

283

Naringenin

++

++

+++

+

+

+++

e

13 14

9.246 9.584

261.1119 333.1699

1.17 -1.66

C15H16O4 C19H24O5

330 320

Meranzin Marmin

++ +

++ +

++ +

+

+

+ +

d d

15

10.596 217.0495

0.21

C12H8O4

320

Bergaptan*

++

++

+

+

+

+

d

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10.664 261.1126

-1.70

C15H16O4

Isomaranzin *

+++

+++

+++

+

+

++

d

17 18 19

10.934 471.2017 11.140 473.2156 11.474 515.227

-2.28 1.19 0.45

C26H30O8 C26H32O8 C28H34O9

210 210 210

Limonin Isoobacunoic acid Nomilin*

++ + ++

++ ++ ++

+++ + ++

+ +

++ +

+++ +++

f f f

20

12.284 455.2074

-1.87

C26H30O7

210

Obacunone*

+++

+++

+++

-

+

+++

f

21

12.962 271.0965

0.02

C16H14O4

310

Imperatorin

++

++

++

+

++

++

d

+++

+++

+++

+

++

++

d

22

14.039 271.0965

0.02

C16H14O4

#

Unknown

*

Confirmed by standard compounds.

**

320

310

*

Isoimperatorin

The criteria of the grading levels were defined according to the peak areas after re-constructing extracted ion chromatograms of the target

compounds: –, not detected; peak area in the range of (0-1) × 105 defined as “+”, meaning low-level; (1-10) × 105 defined as “++”, meaning moderate-level; peak area > 10 × 105 defined as “+++”, meaning high-abundant. ***

Different letters represent different classes of compounds: a - flavone C-glycosides; b - flavone O-glycosides; c - flavanone O-glycosides; d -

coumarins; e - flavanone aglycone; f - limonoids.

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Table 2. Comparisons of Four Main Compounds in Fruit, Flower, Leaf and Branch of CGT Sample a

a

Naringin b

Rhoifolin

Meranzin hydrate

Isoimperatorin

4.81 ± 0.07

1.25 ± 0.03

2.08 ± 0.02

S1

284.35 ± 1.52

S2

257.06 ± 2.04

8.21 ± 0.12

1.14 ± 0.01

3.96 ± 0.06

S3

251.46 ± 1.87

5.20 ± 0.08

1.20 ± 0.02

1.54 ± 0.01

S4

269.21 ± 1.99

15.04 ± 0.23

0.90 ± 0.01

1.38 ± 0.01

S5

222.01 ± 2.22

20.52 ± 0.21

1.82 ± 0.01

2.69 ± 0.05

S6

258.56 ± 2.16

15.11 ± 0.27

1.37 ± 0.01

1.37 ± 0.01

S7

262.88 ± 3.70

16.14 ± 0.20

1.42 ± 0.01

3.13 ± 0.04

S8

246.30 ± 1.75

17.06 ± 0.19

1.74 ± 0.01

1.27 ± 0.01

M1

57.41 ± 0.59

4.78 ± 0.05

2.17 ± 0.03

1.80 ± 0.03

M2

78.44 ± 1.01

8.13 ± 0.06

1.53 ± 0.01

1.64 ± 0.02

M3

79.81 ± 0.77

7.90 ± 0.05

1.98 ± 0.01

1.63 ± 0.02

M4

62.06 ± 0.80

5.64 ± 0.08

1.76 ± 0.01

1.38 ± 0.02

M5

75.46 ± 1.33

9.27 ± 0.10

1.01 ± 0.01

0.89 ± 0.01

M6

86.09 ± 0.88

9.22 ± 0.16

2.07 ± 0.02

1.40 ± 0.01

L1

36.07 ± 0.17

3.26 ± 0.06

1.57 ± 0.01

1.12 ± 0.02

L2

49.88 ± 0.59

5.25 ± 0.04

1.29 ± 0.01

1.07 ± 0.01

L3

39.64 ± 0.41

4.99 ± 0.06

0.73 ± 0.01

0.71 ± 0.005

L4

45.12 ± 0.73

4.96 ± 0.08

1.53 ± 0.01

0.64 ± 0.004

L5

43.24 ± 0.55

5.19 ± 0.04

1.25 ± 0.02

1.16 ± 0.01

L6

44.32 ± 0.71

4.92 ± 0.02

0.95 ± 0.01

1.91 ± 0.02

c

0.13 ± 0.01

F1

167.38 ± 0.92

1.30 ± 0.01

-

F2

117.94 ± 1.56

1.08 ± 0.01

-

0.11 ± 0.00

F3

121.94 ± 1.30

1.12 ± 0.02

-

0.11 ± 0.00

Leaf

1.93 ± 0.01

1.87 ± 0.02

-

-

Branch

5.24 ± 0.05

0.10 ± 0.00

-

0.03 ± 0.00

S1-8 represents small fruit; M1-6 represents medium fruit; L1-6 represents

large fruit; F1-3 represents flower. b

Values are expressed as mean ± SD (n=3) in mg/g of dried weight.

c

Less than the limit of quantitation.

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Figure graphics Figure 1

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