全 文 ：第 49 卷 第 10 期
2 0 1 3 年 10 月
林 业 科 学
SCIENTIA SILVAE SINICAE
Vol. 49，No. 10
Oct．，2 0 1 3
doi:10．11707 / j．1001-7488．20131020
Received date: 2012 － 07 － 13; Revised date: 2013 － 05 － 06．
Foundation project: This research was supported by the State Forestry Administration，P． R． China 948 Project (2009-4-61) ; the National Science
and Technology Infrastructure Program (2012BAD23B03) ; the Basic Science Research Fund Program of the International Centre for Bamboo and Rattan
( ICBR) (12618-6) ．
* Yue Yongde is corresponding author．
3 个属竹叶中黄酮类化合物的比较*
魏 琦 岳永德 汤 锋 孙 嘏
(国际竹藤中心 北京 100102)
摘 要: 采用高效薄层色谱法(HPTLC)检测箣竹属、牡竹属及刚竹属 11 个竹种竹叶中牡荆苷、异牡荆苷、荭草
苷、异荭草苷和苜蓿素。采用自动多级展开法，5 种黄酮类化合物分离效果良好，其 RF值分别为 0. 25，0. 32，0. 38，
0. 47 和 0. 88，回收率在 79. 01% ～ 106. 85%之间。3 属 11 种竹叶中黄酮类化合物种类和含量具有差异，紫竹中 5
种黄酮含量总和最高，为 0. 132% ; 麻竹中 5 种黄酮含量总和最低，为 0. 015%。
关键词: 高效薄层色谱(HPTLC); 竹叶黄酮; 定性及定量分析; 箣竹属; 牡竹属; 刚竹属
中图分类号: S795 文献标识码: A 文章编号: 1001 － 7488(2013)10 － 00127 － 08
Comparison of Flavonoids in the Leaves of Three Genera of Bamboo
Wei Qi Yue Yongde Tang Feng Sun Jia
( International Center for Bamboo and Rattan Beijing 100102)
Abstract: Isoorientin，orientin，isovitexin，vitexin and tricin in the leaves of three genera 11 bamboos been compared
by a simple high-performance thin-layer chromatographic (HPTLC) method． The multistage development was performed in
Automated Multiple Development (AMD2) and solvents with different ratios were used as mobile phase． The developed
plates were scanned by TLC Scanner3． The components were separated well and R F of the five flavonoids 0. 25，0. 32，
0. 38，0. 47 and 0. 88 respectively． The method was validated for precision，accuracy，selectivity and repeatability，and
expressed as coefficient of variation ( CV ［%］) ． The kinds of flavonoids and the concentrations in the eleven species
bamboo of three different genera were significantly different． Phyllostachys nigra the maximum total flavonoids of 0. 132%，
and the minimum of 0. 015% ． The results would be useful in extracting natural bamboo-leaf flavonoids．
Key words: HPTLC; bamboo-leaf flavonoids; qualitative and quantitative analysis; Bambusa; Dendrocalamus;
Phyllostachys
Being a renewable natural resource and a viable
replacement for wood，bamboo is a vital component of
forest resources of the world ( Jiang，2007) ． Recently，
bamboo resource utilizations are drawing an ever
increasing attention from different countries of the world
(Yue et al．，2007) ． Many species of bamboo can be
the medicinal plants because of the active components
they have (China national group corp of traditional ＆
herbal medicine， 1994 ) ． Bamboo-leaf flavonoids，
important components of plant flavones，have a variety
of medical and biological activities ( Jing，2009) ． For
instance，they are effective in anti-free radical，anti-
oxidation，anti-aging，anti-bacteria，anti-inflammatory
applications (Wang et al．，2007; Yang et al．，2011)，
and can be used to regulate blood fat，prevent the
cardio-cerebrovascular diseases (Liu et al．，2009) ． As
functional components， bamboo-leaf flavonoids also
have the utilization in manufacturing various products
such as cosmetic，feed additive ( Zhang et al．，2004;
Feng et al．， 2011; Zhang et al．， 2003 ) ． The
comparison of flavonoids in the leaves of three different
genera of bamboo is useful for choosing bamboo species
to extract natural bamboo-flavonoids and routine quality
control of the leaves of different bamboo species． Many
methods such as spectrophotometry ( SP )， high
performance liquid chromatography ( HPLC )， high
林 业 科 学 49 卷
performance thin layer chromatography (HPTLC) have
been reported for the study of bamboo-leaf flavonoids
( Guo et al．，2007; Wang et al．，2010; Sun et al．，
2010; Cui et al．， 2011 ) ． HPTLC is simpler，
inexpensive，highly efficient and accurate，and it has
the ability to analyze 10 to 20 samples simultaneously
using a small quantity of organic solvent (Wang et al．，
2010; Sherma，2002) ． The purpose of this paper was
to compare the flavonoid compounds in three genera of
bamboo by HPTLC (Cui et al．，2011) ．
1 Materials and methods
1. 1 Materials，reagents，solvents and absorbents
Eleven bamboos from three genera were studied，
namely Bambusa textilis，B． tuldoides，B． chungii and
B． multiplex; Dendrocalamus giganteus，D． minor and
D． latiflorus; Phyllostachys glauca，P． heteroclada，
P． nigra and P． nidularia． These eleven bamboos
leaves were collected at random from Kunming of
Yunnan，Yongan of Fujian，Changning of Sichuan，
Nanjing of Jiangsu and Nanchang of Jiangxi in China．
The flavonoid standards isoorientin， orientin，
isovitexin，vitexin and tricin ( Purity ＞ 98%，HPLC)
were purchased from Shanghai Winherb Medical
Science Co．， Ltd， Shanghai， China． Aluminum
trichloride was purchased from Tianjin Guangfu Fine
Chemical Research Institute，Tianjin，China． Acetone，
methanol，ethyl acetate，methylene chloride， formic
acid and ethanol were analytical grade，purchased from
Beijing Chemical Works ( Beijing， China ) and
Sinopgarm Chemical Reagent Co．， Ltd ( Beijing，
China) ． Water was purified with an ultrapure water
system (Purelab Plus; Pall Life Sciences，Ann Arber，
MI) ．
Chromatography was performed on HPTLC plates
precoated with silica gel HPTLC-Fertigplatten Nano-
DURASIL － 20 UV254(10 cm × 20 cm，0. 2 mm layer，
product No． 812014，Macherey-Nagel，Germany) ．
1. 2 Instruments
Hamilton syringe ( Bonaduz， Switzerland )，
Linomat5 applicator ( CAMAG， Switzerland )，
Automated Multiple Development ( AMD2，CAMAG，
Switzerland)，TLC Scanner3 (CAMAG，Switzerland)
and Reprostar3 (CAMAG，Switzerland) were used in
this study．
1. 3 Standard solutions preparation
The stock solution of orientin was prepared by
dissolving 5 mg orientin in 7 mL methanol-ethanol-
water 2∶ 3∶ 2 ( v / v) in a 10 mL volumetric flask and
diluting to volume with methanol． The stock solution of
vitexin was prepared by dissolving 5 mg vitexin in 6 mL
methanol-ethanol-water 1∶ 2∶ 3 ( v / v ) in a 10 mL
volumetric flask and adjusting to volume with
methanol． The stock solution of isoorientin，isovitexin，
tricin were prepared in the same way，by dissolving
5 mg isoorientin， isovitexin， tricin respectively in
methanol in 10 mL volumetric flasks．
The mixed standard solution of the five flavonoids
was prepared by mixing 0. 9 mL isoorientin，orientin，
isovitexin，vitexin and tricin stock solution respectively
in a 5 mL volumetric flask，and stored at 4 ℃ ．
1. 4 Sample preparation
Bamboo leaves were dried in shade and samples
(20 g) were extracted with 95% ( v / v) ethanol-water
(3 × 250 mL; each time for 0. 5 h ) by ultrasonic
extraction． The extracts was obtained after removing
the solvent by vacuum distillation， suspended in
1 000 mL water respectively， and extracted with
1 000 mL petroleum ether in separation funnels． After
being extracted with petroleum ether，the fraction was
subjected to chromatography on a 50 cm × 3 cm column
packed with polyamide ( Taizhou Si-jia Biochemical
Plastic Company，Zhejiang，China) and eluted with
50% ( v / v) ethanol-water． The eluent was collected
and enriched in vacuum distillation to give the crude
flavonoids of bamboo leaves． The crude flavonoids
samples of the eleven bamboo species were made
respectively． Sample solutions were prepared by
dissolving 50 mg crude flavonoids in 5 mL methanol-
ethanol-water 2∶ 1∶ 2 ( v / v) in a 5 mL volumetric flask．
1. 5 Chromatography
After being cleaned with methanol and methylene
chloride to remove the impurity in the precoat， the
HPTLC plates were activated at 105 ℃ ． Solutions were
applied as 7. 0 mm bands by Linomat 5 applicator with
a 100 μL Hamilton syringe． Multistage development
was performed in AMD2． The plate was developed in
the first step with methanol-ethyl acetate-methylene
chloride 20∶ 35∶ 45 ( v / v) to 50 mm; in the second
step with acetone-methanol-ethyl acetate-methylene
821
第 10 期 魏 琦等: 3 个属竹叶中黄酮类化合物的比较
chloride 5 ∶ 10 ∶ 35 ∶ 50 ( v / v) to 75 mm; in the third
step with acetone-methanol-ethyl acetate-methylene
chloride 10 ∶ 5 ∶ 30 ∶ 55 ( v / v) to 90 mm ( Tab． 1 ) ．
Formic acid was added to each components of the
mobile phase，and the concentration of formic acid in
each components is 10% ( v / v) ． The developed plate
was dried in air and sprayed with 1% ( w /w )
aluminum trichloride in ethanol as the chromogenic
reagent． Then the plate was left for derivatization and
scanned at 366 nm with TLC Scanner3 controlled by
Wincats software． Peak areas were recorded and the
images of plates were documented by Digistore 2
software．
Tab． 1 Process of the development
Process
Development
distance /mm
Acetone Methanol
Solvents
(%，v / v)
Ethyl
acetate
Methylene
chloride
StepⅠ 50 0 20 35 45
StepⅡ 75 5 10 35 50
StepⅢ 90 10 5 30 55
1. 6 Quantification of the flavonoids
A series of standard solutions of isoorientin，
orientin，isovitexin，vitexin and tricin (90，175，350，
700， 1 050， 1 400， 1 750 ng per bands ) were
prepared to construct standard curve by replicate (n =
3) plotting peak area against the amount of flavonoid．
The quantification of the flavonoids in different samples
was calculated．
1. 7 Validation of the method
Instrument precision was checked by replicate
(n = 10) scanning the bands of isoorientin (270 ng)，
orientin (270 ng)，isovitexin (270 ng)，vitexin (270
ng) and tricin (540 ng) ten times and was expressed
as coefficient of variation ( CV ［%］) ． The
repeatability of standards was assessed by replicate
( n = 9 ) analysis of 180 ng bands obtained from
standard solutions of isoorientin，orientin，isovitexin，
vitexin and tricin，expressed as coefficient of variation
( CV ［%］) ． To test the repeatability of sample
application which expressed as coefficient of variation
(CV ［%］)，3，4， and 5 μL of each of sample
solution was applied in triplicate to a plate and the
peak areas were obtained at last． For accuracy testing
this experiment was repeated three times． Limits of
detection for the flavonoids were measured by reducing
the amounts of the standard solutions applied to the
plates．
The intraday precision was studied by scanning a
series concentration ( 90， 135， 180 ng ) of the
standards bands for six times on the same day (2nd h，
4 th h，6 th h，8 th h，10 th h，12 th h ) and the interday
precision was studied by scanning a series of
concentration ( 90，135，180 ng ) of the standards
bands for eight times on different days (1 st，2nd，3 rd，
4 th，5 th，6 th，7 th，8 th day)，which were expressed as
coefficient of variation (CV［%］) ．
The accuracy of the method was tested by
obtaining recovery at three concentrations ( addition to
a preanalyzed sample of of 50%，100% and 150% of
the amount present in sample ) ． Average recovery
(% ) was calculated．
2 Results and discussion
2. 1 Chromatography
Silica was selected to be the absorbent in this
study． The standard components in samples are
separated well and resolved from other components and
can be quantified． A suitable resolution of the five
flavonoids standards ( Isoorientin， RF = 0. 25，
Orientin，RF = 0. 32，isovitexin，RF = 0. 38，vitexin，
RF = 0. 47，tricin，RF = 0. 88) is obtained． A typical
chromatography from the mixed standards is shown in
Fig． 1．
Fig． 1 Absorption spectrogram obtained from the mixed
standards of isoorientin，orientin，isovitexin，vitexin and
tricin，scanned at 366 nm
The identification was performed by detecting the
UV absorption spectra of the standards and which in
the samples extracted from bamboo leaves，and the
bands from samples were confirmed with those in mixed
standards． The fingerprint of the eleven bamboo
species was shown in Fig． 2．
921
林 业 科 学 49 卷
Fig． 2 Fingerprint photograph of UV366 detection of bamboo-leaf-flavonoids
1 － 3，B． textilis; 4 － 6，B． tuldoides; 7 － 9，B． chungii; 10 － 12，B． multiplex; 13 － 15，D．
giganteus; 16 － 18，D． minor; 19 － 21，D． latiflorus; 22 － 24，P． glauca; 25 － 27，P． heteroclada;
28 － 30，P． nigra; 31 － 33，P． nidularia; A-I，Standards．
2. 2 Validation
The method was validated for linearity，precision，
repeatability，sensitivity and accuracy (Tab． 2 and 3) ．
The relationships between peak area and amount of the
flavonoid were found to be linear in the range of 90 to
1 750 ng per bands． Results from determination of the
recovery of the flavonoids at three levels from the
different bamboo species are presented in Tab． 4．
Tab． 2 Method validation data for quantification of the five flavonoids
Property of the method Isoorientin Orientin Isovitexin Vitexin Tricin
Instrument precision (CV［%］，n = 10) 0. 70 0. 72 0. 52 0. 68 0. 75
Repeatability of standards(CV［%］，n = 9) 1. 87 1. 19 0. 96 0. 85 1. 28
Repeatability of samples(CV［%］，n = 9) 1. 94 0. 87 0. 93 1. 02 1. 36
Linearity 0. 987 3 0. 988 4 0. 997 5 0. 988 8 0. 974 2
Equation
y = 13. 32x +
1 267. 7
y = 10. 16x +
293. 04
y = 13. 39x +
1 262. 7
y = 9. 14x +
698. 07
y = 7. 11x +
2 033. 7
Limit of detection / ng 35 40 25 25 25
Tab． 3 Results of the intraday and interday precision
Components
Concentration /
［ng per band］
Intraday
precision
(CV［%］)
interday
precision
(CV［%］)
Isoorientin 90 1. 26 1. 90
135 1. 00 1. 77
180 1. 87 1. 74
Orientin 90 1. 50 1. 96
135 1. 45 1. 61
180 1. 69 1. 86
Isovitexin 90 1. 75 1. 54
135 1. 29 0. 93
180 1. 11 1. 42
Vitexin 90 1. 29 1. 87
135 1. 84 1. 94
180 1. 43 1. 96
Tricin 90 1. 54 1. 57
135 1. 95 1. 95
180 1. 66 1. 80
2. 3 Determination of flavonoids in eleven bamboo
species
The bamboo-leaf flavonoid sample extracts from the
eleven different bamboo species were compared by use
of the proposed method． The absorption spectrograms at
366 nm of the samples are shown in Fig． 3． The
quantification of flavonoids in bamboo samples are
presented in Tab． 5． In the eleven species from three
bamboo genera，Phyllostachys nigra (Lodd． ex Lindl． )
Munro the maximum of 0. 132%，and the minimum
of 0. 015% ．
031
第 10 期 魏 琦等: 3 个属竹叶中黄酮类化合物的比较
Fig． 3 Absorption spectrogram at UV366 of the three bamboo genera
A: B． textilis; B: B． tuldoides; C: B． chungii; D: B． multiplex; E: D． giganteus; F: D． minor; G: D． latiflorus; H: P． glauca; I: P．
heteroclada; J: P． nigra; K: P． nidularia．
3 Conclusions
Flavonoids in the leaves of three different bamboo
genera have been compared by a densitometric HPTLC
method． The results would be useful for choosing
bamboo species to extract natural bamboo-flavonoids
and routine quality control of the leaves of different
bamboo species． The HPTLC method was simple，
precise，efficient，sensitive and accurate，and it could
be useful for fingerprint studies and for quality control
of bamboo-leaf-flavonoids and their products．
The results obtained reveal differences in the
flavonoid composition of the different bamboo species
from different bamboo genera． The in P． nigra was the
maximum in the eleven bamboo species． It was
established that isoorientin and isovitexin were the
common compounds in the bamboo species tested．
Orientin was found in the extracts of D． giganteus，D．
131
林 业 科 学 49 卷231
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.1
3
第 10 期 魏 琦等: 3 个属竹叶中黄酮类化合物的比较 331
Ta
b．
5
Q
ua
nt
ifi
ca
tio
n
of
fiv
e
fla
vo
no
id
s
in
ba
m
bo
o-
le
af
-f
la
vo
no
id
sa
m
pl
es
de
te
rm
in
ed
by
H
PT
LC
m
et
ho
d
in
th
e
el
ev
en
ba
m
bo
o
sp
ec
ie
s①
Ba
m
bo
o
ge
ne
ra
Ba
m
bo
o
sp
ec
ie
s
Is
oo
rie
nt
in
［
%
，
w
/w
］
Or
ie
nt
in
［
%
，
w
/w
］
Is
ov
ite
xi
n
［
%
，
w
/w
］
Vi
te
xi
n
［
%
，
w
/w
］
Tr
ic
in
［
%
，
w
/w
］
AS
AL
AS
AL
AS
AL
AS
AL
AS
AL
TF
L
［
%
，
w
/w
］
Ba
m
bu
sa
B．
tex
til
is
5.
00
9
±
0.
14
a
0.
04
2
ND
ND
2.
09
7
±
0.
06
a
0.
01
7
ND
N D
ND
ND
0.
05
9
B．
tu
ld
oi
de
s
1.
91
3
±
0.
08
f
0.
04
3
ND
ND
0.
40
6
±
0.
05
f
0.
00
9
ND
ND
ND
ND
0.
05
2
B．
ch
un
gi
i
2.
80
0
±
0.
08
e
0.
02
0
ND
ND
1.
39
3
±
0.
05
c
0.
01
0
ND
ND
ND
ND
0.
03
0
B．
m
ul
tip
lex
1.
63
2
±
0.
08
g
0.
01
1
ND
ND
0.
69
2
±
0.
04
d
0.
00
5
ND
ND
ND
ND
0.
01
6
De
nd
ro
ca
la
m
us
D
．
gi
ga
nt
eu
s
2.
96
8
±
0.
01
d
0.
06
7
0.
58
5
±
0.
06
c
0.
01
3
0.
53
1
±
0.
02
e
0.
01
2
0.
47
1
±
0.
02
a
0.
01
1
ND
ND
0.
10
3
D
．
m
in
or
1.
01
0
±
0.
03
i
0.
02
4
ND
ND
0.
22
0
±
0.
01
h
0.
00
5
ND
ND
ND
ND
0.
02
9
D
．
la
tif
lo
ru
s
0.
11
2
±
0.
03
j
0.
00
2
0.
20
4
±
0.
03
d
0.
00
4
0.
31
7
±
0.
03
g
0.
00
6
0.
13
3
±
0.
02
b
0.
00
3
ND
ND
0.
01
5
Ph
yl
lo
sta
ch
ys
P．
gl
au
ca
3.
39
6
±
0.
07
b
0.
07
1
1.
52
0
±
0.
01
a
0.
03
2
0.
66
3
±
0.
03
d
0.
01
4
ND
ND
0.
24
5
±
0.
08
a
0.
00
5
0.
12
2
P．
he
ter
oc
la
da
1.
23
3
±
0.
05
h
0.
02
0
ND
ND
0.
72
4
±
0.
09
d
0.
01
2
ND
ND
ND
ND
0.
03
2
P．
ni
gr
a
2.
78
4
±
0.
06
e
0.
08
7
1.
23
3
±
0.
02
b
0.
03
8
0.
22
0
±
0.
01
h
0.
00
7
0.
00
8
±
0.
00
3c
0.
00
03
ND
ND
0.
13
2
P．
ni
du
la
ria
3.
23
0
±
0.
09
c
0.
06
4
ND
ND
2.
00
1
±
0.
09
b
0.
04
0
ND
ND
ND
ND
0.
10
4
①
M
ea
n
±
SD
;
a
to
jm
ea
n
th
e
di
ffe
re
nc
es
be
tw
ee
n
th
e
sp
ec
ie
s
af
te
r
D
un
ca
n
va
ria
nc
e
an
al
ys
is
;
A
S:
A
m
ou
nt
in
cr
ud
e
fla
vo
no
id
sa
m
pl
es
;
A
L:
A
m
ou
nt
in
dr
y
ba
m
bo
o
le
av
es
;
TF
L:
In
dr
y
ba
m
bo
o
le
av
es
;
N
D
:
N
o
de
te
ct
ed
．
林 业 科 学 49 卷
latiflorus，P． glauca and P． nigra，while vitexin was
found in the species of D． giganteus，D． latiflorus and
P． nigra，tricin was only found in the extracts of P．
glauca． The results that some flavonoids were not
found in some extracts of bamboo species might
because of the incomplete extraction，low concentration
of the flavonoids or the limits of detection．
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(责任编辑 王艳娜 石红青)
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