Molecules 2010, 15, 5378-5388; doi:10.

3390/molecules15085378

OPEN ACCESS

molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article

Simultaneous Determination of Flavonoids in Different Parts of

Citrus reticulata ‘Chachi’ Fruit by High Performance Liquid
Chromatography—Photodiode Array Detection
Yinshi Sun 1, Jianhua Wang 1,*, Shubo Gu 1, Zhengbo Liu 2, Yujie Zhang 2 and Xiaoxia Zhang 2
1
State Key Laboratory of Crop Biology, College of Agronomy, Shandong Agricultural University,
Taian, 271018, China
2
Agricultural Science Research Institute of Shandong, Taian, 271000, China

* Author to whom correspondence should be addressed; E-Mail: sdauwangjh@163.com;

Tel.: +86 538 8242226; Fax: +86 538 8242226.

Received: 12 July 2010; in revised form: 27 July 2010 / Accepted: 2 August 2010 /
Published: 5 August 2010

Abstract: Flavonoids are important polyphenolic secondary metabolites in plant. Citrus

reticulata ‘Chachi’ fruit are rich in flavonoids and are being used as functional antioxidant
ingredients for the treatment of atherosclerosis and cancer, etc. A high performance liquid
chromatography-photodiode array detection system was used to analyze five flavonoids,
namely, naringin, hesperidin, didymin, tangeretin and nobiletin, in different parts of C.
reticulata ‘Chachi’ fruit. The chromatographic analysis was performed on a C18 column
with a gradient elution of acetonitrile and water at a flow rate of 1.0 mL/min. Detection
was carried out using a photodiode array detector at 280 nm. The calibration curves for the
determination of all analytes showed good linearity over the investigated ranges
(R2 > 0.9995). Precision and reproducibility were evaluated by six replicated analyses, and
the R.S.D. values were less than 0.9% and 2.7%. The recoveries were between 98.37 and
103.89%. This method is promising to improve the quality control of different parts of C.
reticulata ‘Chachi’ fruit.

Keywords: high performance liquid chromatography–photodiode array detector

(HPLC–PDA); quantitative analysis; flavonoids; Citrus Reticulata ‘Chachi’ fruit;
simultaneous determination
Molecules 2010, 15 5379

1. Introduction

Citrus reticulata ‘Chachi’ is a general genus and species (i.e. taxon) of tangerines [1,2]. Its dried and
mature peel Pericarpium Citri Reticultae (Guang Chenpi) has been recorded in the Chinese Pharmacopoeia
as appropriate for medical use [3]. C. reticulata ‘Chachi’ are also consumed as culinary seasonings and tea
ingredients in China [4]. Now, pharmacological research has indicated that Pericarpium Citri Reticultae
exhibits significant antimutagenic [5], antiinflammatory [6,7], antioxidant [8,9], antitumor [10,11], and
antiatherosclerosis [12,13] functions and reduces phlegm in the lung [3]. It is well known that various
flavonoids including naringin, hesperidin, didymin, tangeretin and nobiletin (structures shown in Figure 1)
are the main bioactive constituents of Pericarpium Citri Reticultae. Recently, more attention had been paid
to flavonoids and some publications have suggested they might play important roles in anticancer
activity [14–16].
So far, quite a few approaches have been developed for the determination of the bioactive constituents
from C. reticulata ‘Chachi’, including many studies on flavonoids from different citrus species and citrus
juices [17–22], but there is no systematic study on simultaneous determination of five flavonoids in
different parts of C. reticulata ‘Chachi’. The purpose of this work was to determine and analyse five
flavonoids in the parts of peel, pith, endocarp, pulp and seeds of C. reticulata ‘Chachi’ fruit by HPLC,
which would be useful for quality control applications to citrus and other plantd associated with
these ingredients.

Figure 1. Chemical structures of the five flavonoids.

H
O

O
C
H
3
O
H
O H

H
C
O

O
O

3
O
H

O
C
H
O

3
H H
C
3O

H
C
O
H
O

3
O
H

O
H

O
C
H

O
3
H
O
C
H

naringin tangeretin
3

O
H
O

O
O O H
H

O
H
O
H

O
O

O
C
H
O
H

3
H
O

hesperidin
C
H
3

O
C
H

O
C
H
3

3
O
H

O
H
O

O
O O H
H

H
C
O

O
O

3
H

O
C
H
3
O

O
C
H
O
H

H
C
O
3

O
C
H

O
O
H

didymin nobiletin
Molecules 2010, 15 5380

2. Results and Discussion

2.1. Optimization of the solvent to solid ratio

Generally, a larger solvent volume can dissolve constituents more effectively, leading to an
enhancement of the extraction yield [23]. However, this will lead to excess work in the concentration
process, and causing a waste of solvent. On the other hand, addition of a small amount of solvent will
result in the lower yields of the target constituents [24]. In this study, the solvent to solid ratio was
investigated in the range of 10–35 mL/g. As shown in Figure 2, by increasing the solvent to solid ratio,
the extraction yields were increased, but when the solvent to solid ratio increased over 20 mL/g, there
are no significant differences. Eventually, 20 mL/g was selected as the optimum solvent to solid ratio.

Figure 2. Effect of solvent to solid ratio on the yield of flavonoids (other conditions were
fixed at time = 60 min, temperature = 30 °C, power = 250 W).

1.6
6
1.4
1.2 5

1 4
Yield (%)

0.8
Naringin 3
0.6 Didymin
Tangeretin 2
0.4
Nobiletin
0.2 Hesperidin 1

0 0
10 15 20 25 30 35
Solvent to solid ratio (mL/g)

2.2. Optimization of the extraction time

Figure 3 shows the effects of extraction time on the yield of naringin, hesperidin, didymin,
tangeretin and nobiletin. The results indicated that the changing trends of extraction yields of the five
target compounds were consistent on the whole. When the extraction time was increased up to 60 min,
the increase of the yields were obviously enhanced, but then they leveled off and did not change
significantly. Considering that shorter extraction times could cause incomplete extraction and longer
extraction timea could be time and solvent wasting, eventually, 60 min was selected as the optimal
extraction time.
Molecules 2010, 15 5381

Figure 3. Effect of extraction time on the yield of naringin, hesperidin, didymin, tangeretin
and nobiletin (other conditions were fixed at solvent to solid ratio = 20, temperature = 30 °C,
power = 250 W).

1.6
6
1.4
1.2 5

1 4
Yield (%)

0.8
Naringin 3
0.6 Didymin
Tangeretin 2
0.4
Nobiletin
0.2 Hesperidin 1

0 0
10 20 40 60 80 100 120
Time (min)

2.3. Optimization of the wavelength

The PDA UV spectra of naringin, hesperidin, didymin, tangeretin and nobiletin were compared in
the range of 210–400 nm (Figure 4). The results indicated that naringin (Figure 4A) and hesperidin
(Figure 4B) have similar spectra to didymin (Figure 4C), with high absorptions at about 210–227 nm
and 283–285 nm. Tangeretin (Figure 4D) showed maximum responses at the wavelengths of 210 nm,
250 nm, 270 nm and 334 nm. Nobiletin (Figure 4E) showed maximum responses at the wavelengths of
210 nm, 271 nm and 324 nm. Although the five components all presented high absorptions in the
210–227 nm range, due to the end absorption of the elution solvent, the baseline of each spectrum was
not stationary. Finally, we selected 280 nm as the detection wavelength to eliminate the interference of
the elution solvent and to maintain the stability of the determinations.

Figure 4. Photodiode array UV spectra: (A) naringin, (B) hesperidin, (C) didymin, (D)
tangeretin, (E) nobiletin.

A B
214.0
0.50
0.30

0.40 0.25

283.6 0.20
0.30 284.8
AU

AU

0.15
0.20
0.10

0.10
331.1 0.05

0.00 0.00

220.00 240.00 260.00 280.00 300.00 320.00 340.00 360.00 380.00 220.00 240.00 260.00 280.00 300.00 320.00 340.00 360.00 380.00
nm nm
Molecules 2010, 15 5382

Figure 4. Cont.

C D
226. 9
0.70

0.25
0.60

0.20 0.50 334.7

284.8
0.40 250.5
0.15
AU

AU
270.6
0.30
0.10
0.20

0.05
331.1 0.10

0.00 0.00

220.00 240.00 260.00 280.00 300.00 320.00 340.00 360.00 380.00 220.00 240.00 260.00 280.00 300.00 320.00 340.00 360.00 380.00
nm nm

E
0.50

0.45 324.0

0.40

0.35
271.7
0.30
AU

0.25

0.20

0.15

0.10

0.05

0.00

220.00 240.00 260.00 280.00 300.00 320.00 340.00 360.00 380.00

nm

2.4. Optimization of HPLC method

In the present work, a HPLC method for the analysis of the crude sample was established first. In
order to select an appropriate elution system for the HPLC separation of crude samples, different kinds
of solvents (methanol–water, acetonitrile–water) were employed to analyze these. The results indicated
that when acetonitrile–water was used as the mobile phase, major peaks can be obtained and each peak
achieved baseline separation. Besides, the separation conditions of the analytes were optimized by
systematically adjusting the acetonitrile content in the mobile phase. Figure 5A shows the standard
substances with retention times of 6, 7, 20, 31 and 33 min for naringin, hesperidin, didymin, tangeretin
and nobiletin, respectively. Figures 5B–F are the HPLC chromatograms of peel, pith, endocarp, pulp
and seeds of C. reticulata ‘Chachi’ fruit.
Molecules 2010, 15 5383

Figure 5. The HPLC chromatograms of the standard mixture solutions and samples. (A)
standard mixture solutions, (B) peel, (C) pith, (D) endocarp, (E) pulp, (F) seed. Peaks 1, 2, 3,
4 and 5 correspond to naringin, hesperidin, didymin, tangeretin and nobiletin, respectively.
0.40
0.14

0.35
1 A 2 B
0.12

0.30
4 5 0.10
0.25

2 3
0.08
0.20 4

AU
AU

0.15
0.06
5
0.04
0.10

0.05
0.02 1 3
0.00
0.00

0.00 5.00 10.00 15.00 20.00 25.00 30.00 35.00 0.00 5.00 10.00 15.00 20.00 25.00 30.00 35.00
min min

0.20

C
0.14
0.18
D
0.16 1 0.12
1 2
0.14 2 0.10
0.12
0.08
0.10
3
AU
AU

0.08 0.06

0.06 3 0.04
0.04

0.02 4 5
0.02
4 5
0.00 0.00

0.00 5.00 10.00 15.00 20.00 25.00 30.00 35.00 0.00 5.00 10.00 15.00 20.00 25.00 30.00 35.00
min min

0.070
0.050

0.045
1 2 E 0.060 F
0.040
0.050
0.035

0.030 0.040 2
0.025
AU

3
AU

0.030
0.020

0.015 0.020 1
0.010

4 5 0.010
3 4 5
0.005

0.000 0.000

-0.005
0.00 5.00 10.00 15.00 20.00 25.00 30.00 35.00 0.00 5.00 10.00 15.00 20. 00 25.00 30.00 35.00
min min

2.5. Preparation of calibration curve

A series of standard solutions with six different concentrations were analyzed in triplicate by the
established method with injection volumes of 20 μL. The calibration curve for each compound was
constructed by plotting the peak area (y) versus the concentration of standard analyte (x, mg/mL). The
linear regression equations, correlation coefficients (R2) and linearity ranges are given in Table 1.
The limit of detection (LOD) and limit of quantification (LOQ) were the concentrations of a
compound at which its signal-to-noise ratios (S/N) were detected as 3:1 and 10:1, respectively. The
values are also given in Table 1.
Molecules 2010, 15 5384

Table 1. Linear regression equation, correlation coefficient and linearity of five flavonoids.
Linearity LOD LOQ
Compound Regression equation R2
range (μg/mL) (ng/mL) (ng/mL)
naringin y = 212205 x - 140.64 0.9996 3.8-121.6 25.3 76.0
hesperidin y = 240262 x - 144.7 0.9997 3.1-99.2 20.6 62.0
didymin y = 403272 x - 620.05 0.9996 2.0-64.0 13.3 40.0
tangeretin y = 285301 x + 40.826 0.9999 2.3-73.6 18.0 53.9
nobiletin y = 345873 x + 44.507 0.9999 2.1-67.2 16.4 49.2

2.6. Precision

The precision of the method was determined by the analysis of six consecutive injections using the
same standard mixture solution. The values of relative standard deviations (R.S.D.s.) of the peak areas
for naringin, hesperidin, didymin, tangeretin and nobiletin were 0.9, 0.8, 0.5, 0.3 and 0.7% (n = 6),
respectively.

2.7. Reproducibility test

Reproducibility of peels was evaluated by six replicated analyses. The R.S.D.s. of the contents of
naringin, hesperidin, didymin, tangeretin and nobiletin in six replicated peels were 1.5, 2.3, 0.9, 2.7
and 1.4%, respectively (n = 6).

2.8. Recovery test

In the recovery test, three different concentrations of naringin, hesperidin, didymin, tangeretin and
nobiletin were added to known amounts (80, 100 and 120%) of the pre-analyzed peel samples solution,
and then the spiked samples were analyzed three times (n = 5) by the established HPLC method. The
results are shown in Table 2.

Table 2. Recovery test of the five flavonoids from peel (n = 3).

Total Amount
Quantity Recovery
Components quantity quantity R.S.D.(%)
added % (%)
present (mg) found (mg)
naringin 80 0.66 0.67 102.02 1.53
100 0.80 0.82 102.08 0.58
120 0.95 0.96 101.40 1.53
hesperidin 80 44.22 44.94 101.62 4.16
100 55.24 54.85 99.29 4.04
120 66.31 68.43 103.19 1.53
didymin 80 0.97 0.98 101.37 0.58
100 1.23 1.25 101.36 2.08
120 1.48 1.47 99.55 0.58
tangeretin 80 6.27 6.38 101.81 4.04
100 7.75 7.80 100.65 1.00
120 9.34 9.46 101.28 3.61
Molecules 2010, 15 5385

Table 2. Cont.
nobiletin 80 1.20 1.22 103.89 0.58
100 1.53 1.56 102.18 2.52
120 1.84 1.81 98.37 3.00

2.9. Application

The optimal conditions were applied to the quantitative analysis of naringin, hesperidin, didymin,
tangeretin and nobiletin in different parts of C. reticulata ‘Chachi’ fruit and the results are presented in
Table 3. The chromatograms obtained were shown in Figures 5B–F. The identification of the
investigated compounds was carried out by comparison of their retention time and UV spectra of the
standard compounds.

Table 3. Amount of five flavonoids in C. reticulata ‘Chachi’ Fruit (n = 3).

Contents (μg/g) (mean ± S.D.)
Analyte
naringin hesperidin didymin tangeretin nobiletin
Peel 811.5 ± 18.1 55260.4 ± 802.4 1232.7 ± 21.3 7702.1 ± 80.6 1520.4 ± 40.5
Pith 7083.1 ± 90.7 8538.2 ± 57.6 2228.6 ± 50.8 5.2 ± 0.2 194.2 ± 5.6
Endocarp 3180.2 ± 64.9 1810.8 ± 29.7 1668.6 ± 26.4 1.6 ± 0.0 65.7 ± 2.1
Pulp 584.0 ± 14.2 8369.4 ± 75.1 291.3 ± 7.4 10.7 ± 0.1 17.8 ± 0.3
Seed 79.7 ± 3.3 241.6 ± 9.3 24.9 ± 1.1 3.0 ± 0.1 4.2 ± 0.2

3. Experimental

3.1. Reagents and materials

Acetonitrile used for HPLC was of chromatographic grade (Yongda Chemical Factory, Tianjin,
China), and water used was distilled water. Other organic solvents used were of analytical grade and
purchased from Tianjin Chemical Factory (Tianjin, China). A microporous membrane (φ 13 mm,
0.45 μm) from Tianjin Tengda Filtration Instrument Co. (Tianjin, China) was used. Naringin,
hesperidin, didymin, tangeretin and nobiletin were obtained from the authors’ laboratory, their
structures were fully characterized by chemical and spectroscopic methods (UV, IR, NMR, MS). Their
purities were above 98.0% as judged by HPLC–PDA. Fresh Citrus reticulata ‘Chachi’ fruit was
collected from Guangzhou Province, China, and was identified by Dr. Jianhua Wang (College of
Agronomy, Shandong Agricultural University).

3.2. Instrument and chromatography conditions

The high performance liquid chromatography (HPLC) equipment used was a Waters 600E (USA)
system including a 4-solvent delivery system, 600E start-up kit, a 600 pump, 0–20 mL/min, a 2996
photodiode array detector, an Empower Add-on Single System (China), a 4-chamber in-line degasser
and a 600E controller. The analysis was performed on a Symmetry C18 column (250 mm × 4.6 mm i.d.,
5 μm particle size) at ambient room temperature using a gradient elution of acetonitrile (solvent A) and
water (solvent B) at a flow rate of 1.0 mL/min. A gradient program was used as follows: 22–22% A at
Molecules 2010, 15 5386

0–10 min, 22–61% A at 10–35 min and 61–100% A at 35–40 min, re-equilibration duration between
two individual runs was 15 min. Detection was carried out at 280 nm.

3.3. Sample preparation

The peel, pith, endocarp, pulp, seed of C. reticulata ‘Chachi’ fruit were separated and dried to a
constant weight at 60 °C in a vacuum oven, then pulverized to powder (about 40-mesh) with a
disintegrator. The five parts of C. reticulata ‘Chachi’ fruit ground samples were extracted with
methanol (solvent to solid ratio was 20 mL/g) in an ultrasonic water bath for 60 min. All extractions
were done at ambient room temperature and each extraction was repeated three times. The extracts
were combined and concentrated under reduced pressure. The residue was then reconstituted in
methanol to give appropriate concentration. All solutions were filtered through 0.45 μm membrane
filter before direct injection into the HPLC system.

3.4. Preparation of standard solutions

A mixed standard stock solution was prepared by transferring 3.8 mg/mL of naringin, 3.1 mg/mL of
hesperidin, 2.0 mg/mL of didymin, 2.3 mg/mL of tangeretin and 2.1 mg/mL of nobiletin working
standards into a 10 mL volumetric flask and making up to the mark with methanol. Then, the mixed
solution was diluted step by step with methanol to give six different concentrations of working
standard solutions. All solutions were filtered through 0.45 μm membrane filter prior to analysis.

4. Conclusions

A simple, accurate and reliable analytical method for the simultaneous determination of five major
flavonoids from C. reticulata ‘Chachi’ fruit by HPLC-PDA has been developed. The flavonoids,
naringin, didymin, hesperidin, tangeretin and nobiletin, were determined in different parts of C.
reticulata ‘Chachi’ fruit. The results of the analysis indicated that naringin and didymin were mainly
found in pith and endocarp, and hesperidin, tangeretin and nobiletin mainly stored in peel and pith.
This method showed good linearity, sensitivity and sufficient limit of detection. The evaluation of data
could be useful for screening the optimal condition of extraction of different part of C. reticulata
‘Chachi’ fruit, and also facilitate to provide the critical quality assurance of related extracts of Chinese
medicinal plants.

Acknowledgements

Financial support from the Ministry of Science & Technology of China (No. 2005DKA21000) and
Shandong Agricultural University (No. 23489) are gratefully acknowledged.

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