Research Article

Received: 8 June 2008

Revised: 17 November 2008

Accepted: 7 January 2009

Published online in Wiley Interscience: 11 March 2009

(www.interscience.wiley.com) DOI 10.1002/jsfa.3537

Influence of flowering stage of Lonicera

japonica Thunb. on variation in volatiles
and chlorogenic acid
Li-Mei Wang, Mao-Teng Li, You-Yu Yan, Ming-Zhang Ao, Geng Wu
and Long-Jiang Yu
Abstract
BACKGROUND: The volatile oil of Lonicera japonica Thunb. is an edible natural perfume that is often used in foods, cigarettes
and cosmetics. Chlorogenic acid, a major bioactive component of L. japonica, has received much attention recently owing to its
antiviral, anticancer and anti-inflammatory activities. However, to date, few reports have focused on the variation in volatile oil
and chlorogenic acid at different flowering stages of L. japonica.
RESULTS: Obvious variations in components of the volatile oil were observed at six different developmental stages. The primary
components of the volatile oil were linalool, linalool oxide, geraniol and -terpineol, which reached their highest levels at the
silver flowering stage. The highest levels of chlorogenic acid were found at the second white and complete white flowering
stages.
CONCLUSION: The results of this study indicate that the best times to harvest L. japonica flowers for volatile oil and chlorogenic
acid extraction are the silver flowering stage and the second white or complete white flowering stage respectively.
c 2009 Society of Chemical Industry


Keywords: Lonicera japonica Thunb.; volatile oil; gas chromatography/mass spectrometry; chlorogenic acid

INTRODUCTION

J Sci Food Agric 2009; 89: 953957

industry varies considerably, thus affecting both the quality of

volatile oil and the purity of chlorogenic acid produced. Research
on the optimal harvesting stage would therefore be of economic
importance for this species. However, to date, few reports have
focused on the variation in volatile oil and chlorogenic acid at
different flowering stages of L. japonica.
In this study the variation in volatile oil composition and
chlorogenic acid level at different flowering stages of L. japonica
was evaluated. The results should be helpful in determining the
optimal harvesting stage for volatile oil and chlorogenic acid
production from this species.

MATERIAL AND METHODS

Plant material
Flowers of L. japonica were collected on the campus of Huazhong
University of Science and Technology, Wuhan, China at 10 : 00 on
4 May 2008. The collected flowers were divided into six groups

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Correspondence to: Mao-Teng Li, Institute of Resource Biology and Biotechnology, College of Life Science and Technology, Huazhong University of Science
and Technology, Wuhan 430074, China. E-mail: limaoteng426@163.com
Institute of Resource Biology and Biotechnology, College of Life Science and
Technology, Huazhong University of Science and Technology, Wuhan 430074,
China and Key Laboratory of Molecular Biophysics, Ministry of Education,
Wuhan 430074, China

c 2009 Society of Chemical Industry

953

Lonicera japonica Thunb., which belongs to the family

Caprifoliaceae,1 is a medicinal plant widely used in China. As
a traditional Chinese medicine with a wide spectrum of biological
and pharmacological properties, the flowers of L. japonica have
been used in clinical practice for thousands of years for their
antibacterial, antiviral and antioxidant activitives2,3 and in the
treatment of exopathogenic wind-heat, epidemic febrile diseases,
sores, carbuncles and furuncles.1,4,5 A volatile oil and chlorogenic
acid are reported to be effective components of L. japonica.6 The
volatile oil of L. japonica is an edible natural perfume that is often
used in foods, cigarettes and cosmetics.7,8 Chlorogenic acid, which
is a major bioactive component of the flowers of L. japonica, has
received much attention recently owing to its antiviral, anticancer
and anti-inflammatory activities.9,10 However, the volatile oil and
chlorogenic acid are very expensive, with current average prices
of US$3230/kg and US$2000/kg respectively.
In China, although L.japonica flowers from May to September in the
field,11 the flowering duration of individual plants is usually only
58 days. Previous research has found that the flowering period
can be divided into six stages, i.e. the juvenile bud stage, the third
green stage, the second white stage, the complete white stage,
the silver flowering stage and the gold flowering stage.12 Both the
chemical contents and composition vary depending on the flower
collection time,13 15 while the harvesting index of L. japonica
flowers also shows flowering-dependent characteristics.11,16 At
present, the quality of raw materials purchased by the processing

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Volatile oil and chlorogenic acid extraction

The volatile oil was initially extracted by steam distillation for 6 h,
after which the distillate was further extracted using absolute
ether. The organic layer was dried using anhydrous sodium
sulfate, then filtered and concentrated to obtain the volatile
oil, which was stored at 46 C until further analysis by gas
chromatography/mass spectrometry (GC/MS).17
For chlorogenic acid extraction, fresh flowers of L. japonica were
cut into pieces (5 mm). Aliquots of 0.5 g were mixed with 700 mL
L1 ethanol (solvent/sample ratio 20 : 1 v/w), placed in an ultrasonic
extractor and sonicated for 30 min twice.18,19 After collecting the
supernatant, the remaining filtrate was back-extracted again using
the same volume of fresh solvent. The filtrate was collected and
concentrated to obtain concentrated chlorogenic acid solution.
The combined extracts were concentrated to dryness under
vacuum at 45 C. A 4 mg aliquot of each sample was dissolved in
20 mL of high-performance liquid chromatography (HPLC)-grade
methanol and filtered through a 0.45 m membrane.

Volatile oil analysis

The volatile oil was analysed by GC/MS. A Trace MS2000 GC/MS
system (Finnigan, Silicon Valley, California, USA) equipped with a
mass-selective detector with electron impact ionisation was used.
Briefly, the analytes were separated using an HP-5 MS (Agilent,
Santa Clara, USA) capillary column (30 m 0.25 mm, 0.25 m)
inserted directly into the ion source of the MS system. Helium
(99.999% pure) was used as carrier gas at a flow rate of 1.5 mL
min1 . The splitless mode was used, with the injector temperature
set at 220 C. The oven temperature was programmed to increase
from 60 to 150 C at 5 C min1 and then from 150 to 250 C at
8 C min1 , where it was maintained for 10 min.20 The electron
impact ionisation conditions were an ion energy of 70 eV and a
scanning mass range of m/z 50400 in full-scan acquisition mode.
Each component was identified using the NIST or Wiley 6.0 mass
spectral library and other published mass spectra.21 23

RESULTS
Components of volatile oil at different flowering stages
Measurements of the volatile oil obtained from L. japonica at
different flowering stages revealed that the oil content increased
from the first stage on, reached its highest level of 1.3 g kg1
fresh weight (FW) at the silver flowering stage and then started
to decrease (Fig. 1). Likewise, the average fresh weight increased
with flower development but decreased at the gold flowering
stage (Fig. 1). For an individual fresh flower the highest volatile oil
production reached 0.122 mg at the silver flowering stage.
The chemical components of the volatile oil of L. japonica
flowers at different developmental stages, as analysed by GC/MS
and identified using a mass spectral library, are listed in Table 1.
Huge variations in the volatile oil components at the six different
flowering stages were observed, with more than 40 components
being identified. Of these, 34 components accounted for more
than 85% of the total volatile oil. From Table 1 it can be deduced
that the primary components were linalool, linalool oxide, geraniol,
-terpineol, dibutyl phthalate and 9,12,15-octadecatrienoic acid
methyl ester, whose contents varied with the flowering stage. For
example, geraniol, -terpineol and linalool reached their highest
levels (8.17, 10.57 and 15.35% respectively) at the silver flowering
stage, whereas linalool oxide and 9,12,15-octadecatrienoic acid
methyl ester reached their highest levels at the second white and
third green stages respectively (Table 1).
In addition to the six major components, four additional
components, acetic acid ethyl ester, benzyl benzoate, nonadecane
and hexadecanoic acid, were present at relatively high levels in
the flowers of L. japonica. The level of acetic acid ethyl ester
increased from the first to the sixth flowering stage, whereas
that of hexadecanoic acid decreased. Nonadecane and benzyl
benzoate reached their highest levels in the third green and gold
flowering stages respectively (Table 1).
Another five components, hexane, tetradecanoic acid, 1octadecanol, tetracosanoic acid methyl ester and hexadecane,
were only observed during the last four flowering stages. Trend
analysis indicated that all these components increased initially
and then decreased during the silver or gold flowering stage, with
the exception of 1-octadecanol which increased during the gold
flowering stage (Table 1).
The remaining components, which included 3-hexenol, 2butanol, heptanal and octanone, were only present at one or
two flowering stages (Table 1).

954

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Volatile oil content (mg g1 FW)

1.6

Chlorogenic acid analysis

Chlorogenic acid was analysed by HPLC. An Agilent 1200 HPLC
system with an XDB-C18 chromatographic column (4.6 mm
150 mm, 5 m) (Agilent, Santa Clara, USA) was used for the analysis
of a chlorogenic acid standard and the L. japonica samples.
A 5 mg aliquot of the chlorogenic acid standard was dissolved in
100 mL of HPLC-grade methanol. The solution was filtered through
a 0.45 m membrane. The optimised chromatographic condition
was CH3 OH/H2 O/CH3 COOH 15 : 85 : 0.4 (v/v/v). Densitometric
scanning was performed at = 327 nm. A standard curve
was constructed using known concentrations (1050 g mL1 )
of chlorogenic acid. The regression equation was y = 40.769x +
90.247 (R2 = 0.9994).
Levels of chlorogenic acid were determined by comparing the
results for the samples against the standard curve.

0.12

content of volatile oil

average weiht of one flower

1.4

0.1

1.2
0.08

1
0.8

0.06

0.6

0.04

0.4
0.02

0.2
0

Average weight of one flower (g)

according to the characteristics of the juvenile bud stage (A), the

third green stage (B), the second white stage (C), the complete
white stage (D), the silver flowering stage (E) and the gold flowering
stage (F). The samples (three replicates each) were subjected to
volatile oil and chlorogenic acid extraction for further analysis.

L Wang et al.

Different flowering stages

Figure 1. Volatile oil content () and average weight of one flower (
) of
Lonicera japonica at different flowering stages.

c 2009 Society of Chemical Industry

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Variation in volatiles and chlorogenic acid in Lonicera japonica

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Table 1. Chemical components of volatile oil from Lonicera japonica in different flowering stagesa
Content (%)
Molecular
weight

Molecular
formula

Hydrocarbons
Hexane
Pentadecane
Heptadecane
Nonadecane
Octadecane
2,6,10-Trimethyltetradecane
Triacontane
Hexadecane
Eicosane
5-(Prop-2-enoyloxy)pentadecane

86
212
240
268
254
240
422
226
282
282

12.54

9.08

3.01

2.29
7.54

0.94
0.75

3.05

7.23
5.10
1.27

3.16

4.57

1.41
0.47
1.15
1.45
1.11
1.41
1.48
3.72

3.47
0.58

1.09

1.28

7.81

C6 H14
C15 H32
C17 H36
C19 H40
C18 H38
C17 H36
C30 H62
C16 H34
C20 H42
C18 H34 O2

Alcohols
3-Hexenol
2-Butanol
Linalool
Geraniol
1-Octanol
-Terpineol
5-Octen-1-ol
1-Octadecanol
Phytol

100
74
154
154
130
154
128
270
296

0.15
0.24

0.14

0.31
1.05

0.21

0.09

6.41
7.20
15.12
3.04

4.79
1.02
1.53
0.23

2.41
5.38
3.77
0.47
5.62

3.54
0.47

15.35
8.17
0.62
10.57

3.07
2.15

11.20
3.45

2.51

4.86

C6 H12 O
C4 H10 O
C10 H18 O
C10 H18 O
C8 H18 O
C10 H18 O
C8 H16 O
C18 H38 O
C20 H40 O

Aldehydes
Heptanal
Octadecanal

114
268

1.04

2.58
4.51

C7 H14 O
C18 H36 O

Ketones
Octanone

128

1.47

2.06

C8 H16 O

Esters
Acetic acid ethyl ester
Benzeneacetic acid methyl ester
Docosanoic acid methyl ester
Tetracosanoic acid methyl ester
Hexadecanoic acid methyl ester
Docosanoic acid methyl ester
9,12,15-Octadecatrienoic acid methyl ester
Benzyl benzoate
Dibutyl phthalate

88
150
354
382
298
354
292
212
278

3.14

9.85

14.51
2.08
18.67

6.52

16.73
5.47
10.73

7.10

1.76
1.02
0.24
3.17
2.15
14.20

8.15

2.90
2.43

3.82
1.42
5.47

12.30

1.01
1.12

6.17

10.34

14.85
7.58

0.54

2.67
17.15

C4 H8 O2
C9 H10 O2
C23 H46 O2
C25 H50 O2
C19 H38 O2
C23 H46 O2
C19 H32 O2
C14 H12 O2
C16 H22 O4

Epoxides
Linalool oxide

170

0.09

0.17

6.53

4.76

2.41

5.17

C10 H18 O2

Fatty acids
Tetradecanoic acid
Hexadecanoic acid

228
256

41.72

0.26
28.12

0.54
6.24

1.02
8.75

2.28
6.47

4.08

C14 H28 O2
C16 H32 O2

Compound

A, juvenile bud stage; B, third green stage; C, second white stage; D, complete white stage, E, silver flowering stage; F, gold flowering stage.

Overall, the optimal time to harvest the flowers of L. japonica

for volatile oil extraction would seem to be the silver flowering
stage.

J Sci Food Agric 2009; 89: 953957

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955

Changes in chlorogenic acid at different flowering stages

The HPLC results for the chlorogenic acid standard and the ethanolextracted samples of L. japonica are shown in Fig. 2. Measurements
revealed that the chlorogenic acid content varied at different
flowering stages as follows: third green stage > juvenile bud stage

> second white stage > complete white stage > gold flowering
stage > silver flowering stage (Fig. 3). When the changes in
individual flowers were considered, the average chlorogenic acid
production of each flower was obviously enhanced from the
first to the third flowering stage (from 0.147 to 0.475 mg) and
decreased thereafter (from 42.750 to 36.270 mg). To obtain the
highest amount of chlorogenic acid from flowers of L. japonica,
the optimal time for harvesting would appear to be the second
white or complete white flowering stage.

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L Wang et al.

Figure 2. HPLC chromatograms of (a) chlorogenic acid standard and (b) 700 mL L1 ethanol extract of Lonicera japonica flowers.

Chlorogenic acid content

(mg g1 FW)

12
10
8
6
4
2
0

Different flowering stages

Figure 3. Chlorogenic acid content of Lonicera japonica at different

flowering stages.

DISCUSSION

956

In this study the volatile oil content of L. japonica was measured

at different flowering stages and found to reach its highest
level at the silver flowering stage. The variation in volatile oil
composition was also observed at different flowering stages, with
the results indicating that some chemical components might
accumulate during a particular period as the plant adapts to
changes in its environment.24,25 Previous studies have shown that
age-dependent changes in volatile oil content seem to occur in
some other species. For example, Aidi Wannes et al.26 found that
the essential oil yield varied from 0.003 to 0.11% during fruit
development of Myrtus communis var. italica, while Wang et al.27
reported that the volatile oil content varied greatly at different
flowering stages of Osmanthus fragrans Lour.27
Previous studies have found that the primary components of
the volatile oil of L. japonica are monoterpenes, sesquiterpenes
and fatty acids and their esters.28,29 These compounds were also
observed in the present study, but their variation at different flow-

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ering stages has not been not reported before. Linalool, linalool
oxide, geraniol and -terpineol are closely related to the flavour of
L. japonica volatile oil.30,31 As primary components of the volatile
oil, most of them reached their highest levels at the silver flowering
stage in this study, which means that farmers could acquire a
maximum amount of total and major components of the volatile
oil with the same amount of biomass at a certain stage. Knowledge
of the changes in these valuable chemical components should be
advantageous for farmers in harvesting this traditional Chinese
medicinal plant. Interestingly, the levels of three other components, acetic acid ethyl ester, benzyl benzoate and nonadecane,
were observed to be higher in the gold flowering and juvenile
bud stages of L. japonica, which has not been reported previously.
Chlorogenic acid is another important component of
L. japonica that has received considerable attention owing to
its wide distribution and potential biological functions,32 including antioxidant, antimutagenic, analgesic, antipyretic and
anticarcinogenic activities.33 36 So far, most studies have focused on its extraction18,37 and its structural and pharmacological
properties.38 40 A seasonal analysis revealed that the highest and
lowest contents of chlorogenic acid in flowers occurred during
July and September respectively in Ningxia province in northwest
China;41 in contrast, the highest content of this component has
been observed in September.42 However, there are no previous
studies on the variation in chlorogenic acid content at different
flowering stages. The present study showed that the highest
content of chlorogenic acid occurred at the third green stage.
Considering the variation in fresh weight at different flowering
stages, the best harvesting time for high chlorogenic acid yield
should be the second white or complete white stage.

CONCLUSION
By analysing the volatile oil yield, volatile oil components and
average yield of individual flowers, we conclude that the optimal

c 2009 Society of Chemical Industry

J Sci Food Agric 2009; 89: 953957

Variation in volatiles and chlorogenic acid in Lonicera japonica

harvesting period for the extraction of volatile oil from L. japonica
is the silver flowering stage. On the other hand, the best harvesting
period for the extraction of chlorogenic acid is the second white or
complete white flowering stage. These results should be of great
importance for farmers to determine when to harvest L. japonica
crops according to commercial demand.

ACKNOWLEDGEMENTS
This investigation was supported by the National Science
Foundation of China (30600074), the National Technology Support
Program (2006BAC01A16) and the Opening Foundation of Karst
Dynamics Laboratory, Ministry of Land and Resources. The authors
are grateful to Ms Cheng of the Analytical and Testing Center of
Huazhong University of Science and Technology for her valuable
GC/MS analysis.

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