全 文 ：Ｒeceived:24 January ，2013 Ｒevised:25 April，2013
Foundation items:2011 Anhui Provincial Natural Science College Ｒe-
search Project (NO． KJ2011B165) ;2011 Huangshan University Sci-
ences Ｒesearch Project (NO． 2011xkj003)．
* Corresponding author E-mail:biofood@ jlu． edu． cn
天然产物研究与开发 Nat Prod Ｒes Dev 2013，25:1436-1441，1410
文章编号:1001-6880(2013)10-1436-07
高压脉冲电场辅助提取箬叶总黄酮的工艺优化
金声琅1，2，殷涌光2*
1黄山学院旅游学院，黄山 245021;2 吉林大学生物与农业工程学院，长春 130022
摘 要:为研究高压脉冲电场辅助处理对总黄酮提取效果的影响，以箬叶为试材，找出了其最佳的处理技术参
数。在乙醇浓度、电场强度、脉冲数和料液比 4 个单因素试验的基础上，采用 L9(3
4)正交试验来优化工艺参数。
试验结果表明，料液比是最主要的影响因素，其次分别是电场强度、脉冲数和乙醇浓度。在料液比为 1∶ 40，电场
强度为 20 kV /cm，脉冲数为 8，乙醇浓度为 75%的条件下箬叶总黄酮的提取率最高，达到 4． 89%，比传统提取法
提高了 2． 5 倍以上。试验结果表明，高压脉冲电场辅助提取技术在箬叶总黄酮提取工艺中具有较高的应用价
值，可作为一种新型工艺技术进行推广。
关键词:箬叶;总黄酮;高压脉冲电场;辅助提取
中图分类号:TQ917 文献标识码:A
High Intensity Pulsed Electric Fields-Assisted Extraction of Total
Flavonoids from Indocalamus tessellatus Leaves
JIN Sheng-lang1，2，YIN Yong-guang2*
1Tourism College，Huangshan University，Anhui Huangshan 245041，China;
2College of Biological and Agricultural Engineering，Jilin University，Jilin Changchun 130022，China
Abstract:This study established preliminary conditions for enhancing high intensity pulsed electric fields (HIPEF)-as-
sisted extraction of total flavonoids from Indocalamus tessellatus leaves． Four influencing factors including ethanol con-
centration (35%，45%，55%，65%，75%，85%，and 95%) ，electric field intensity (10，20，30，40，50，60 kV /cm) ，e-
lectric pulse number (0，2，4，6，8，10 and 12)and ratio of material-solvent (1∶ 20，1∶ 30，1∶ 40，1∶ 50，1∶ 60 and 1∶ 70)
were investigated using the L9(3
4)orthogonal test． The ratio of material-solvent was the main factor affecting extraction
result，followed by electric field intensity，pulse number and ethanol concentration． The optimal conditions were deter-
mined as follows:material-solvent ratio of 1∶ 40，electric field intensity of 20 kV /cm，pulse number of 8 and ethanol con-
centration of 75% ． Under these conditions，the extraction yield of total flavonoids was 4． 89% ． HIPEF and conventional
extraction method were compared． The extraction yield of flavonoids using the conventional method was 1． 56%，and the
microwave assisted extraction yield of flavonoids was 1． 92% ． The results suggested that the HIPEF-assisted extraction
process was feasible，and the extraction yield of total flavonoids from I． tessellatus leaves was higher than the conventional
method．
Key words:Indocalamus tessellatus leaves;total flavonoids;high intensity pulsed electric fields;assisted extraction
Introduction
China has the most abundant bamboo resources in the
world，and the chemical constituents of bamboo leaves
are widely used in the manufacturing process of new
pharmaceutical and food products［1］． Bamboo species
of the genus Indocalamus have been used for centuries
because of the applicable leaves，permanent aroma and
health protection ［2］． The Medical Canon in Four Sec-
tions (8th century AD，China)has documented the me-
dicinal properties of I． tessellatus leaves in detail． As
the expanding of peoples knowledge and understanding
of its medicinal value，research and development relat-
ed to extracts of I． tessellatus leaves has been extensive-
ly investigated in recent years ［3］．
Modern medical research results show that flavonoids in
bamboo have the functions of anti-oxidation，anti-ag-
ing，reducing myocardial oxygen consumption，mitiga-
ting coronary artery diseases，improving cerebral vascu-
lar blood flow，softening blood vessels，reducing blood
glucose and blood lipids，increasing the bodys immune
system by eliminating free radicals ［3］． Traditional tech-
nology of extracting flavonoids from natural plants has
some disadvantages such as high cost，low production
quantities associated with complex process ［4］． Various
organic solvents such as alcohols (methanol，ethanol，or
alcohol-water mixtures) ，acetone，and ethyl acetate are
used to extract flavonoids from natural plants for indus-
trial development． However，large amounts of solvent
are present and the extraction steps are time-consuming
and tedious (about 20 h for batch processes)［3］． Sev-
eral improved methods have been developed to extract
flavonoids from natural plants，such as microwave-assis-
ted extraction［5］，ultrasound-assisted extraction ［6］ and
supercritical fluid extraction ［1］．
High intensity pulsed electric fields (HIPEF)has been
widely studied as a non-thermal food preservation
method for food pasteurization process． In recent years，
HIPEF has been used to accelerate the extracting
processes and to improve bioactive compound extrac-
tion［7］． Compared with the conventional methods，
HIPEF-assisted extraction method has many advanta-
ges，such as easy operation，reliable operation，few by-
products，high efficiency，good selectivity ［8］． Many
studies focused on investigating the main components of
I． tessellatus leaves ［1-6］． However，there are few reports
on the HIPEF-assisted extraction of flavonoids． In this
research，the application of HIPEF on the extraction of
flavonoids from I． tessellatus leaves，and the effects of a
set of extraction parameters (including material-solvent
ratio，electric field intensity，pulse number and ethanol
concentration)on extraction efficiency were investiga-
ted． The optimal conditions for the HIPEF-assisted ex-
traction of total flavonoids from I． tessellatus leaves were
determined，which provided reliable experimental and
theoretical basis for the further development and utili-
zation of I． tessellatus leaves as a readily accessible
source of bioactive compounds for pharmaceutical us-
age．
Materials and Methods
Materials
Sample
I． tessellatus leaves (chequer-shaped tessellatus leaves，
collection time:December，2010 and July，2011 respec-
tively，place:Huangshan，China)．
Ｒeagents and chemicals
The reference standard of rutin was purchased from Na-
tional Institute for the Control of Pharmaceutical and
Biological Products (purity ≥ 98%)． Ethanol，sodium
nitrite，aluminum nitrate，sodium hydroxide and other
reagents were of analytical grade obtained from com-
mercial sources．
Instruments and equipments
AEU-210 electronic analytical balance (Xiangyi Bal-
ance Instrument ＆ Equipment Co．，Ltd．) ，FWl00
high-speed universal grinder (Tianjin City Taisite In-
strument Co．，Ltd．) ，SHZ-D(Ⅲ) circulating water
vacuum pump (Gongyi Yingyu Yuhua Instrument Fac-
tory) ，LD5-2A centrifuge (Beijing Medical Centrifuge
Factory) ，and GZX-9140M E digital blast oven
(Shanghai Xunda Industrial)were employed in this re-
search．
HIPEF system consists of a repetitive high-voltage
pulse generator，a coaxial liquid materials treatment
chamber，a fiber-optic temperature sensing instrument，
an oscilloscope and a pump (Fig． 1)． The generator is
home-made and has been described in our previous
study ［7-9］． A cylindrical plastic tube closes with two
polished stainless steel electrodes was used as the con-
tinuous treatment chamber． The distance between elec-
trodes was 0． 15 cm and the electrode area (r = 0． 5
mm)was 7． 85 × 10-3 cm2 ． The circuit configuration
generated sawtooth waveform pulses at different fre-
quencies (1-3000 Hz) ，pulse width (2 μs)and elec-
tric field strengths (1-70 kV /cm)． The actual electric
field strengths and electrical intensity applied were
measured in the treatment chamber with a high-voltage
probe and a current probe respectively connected to an
oscilloscope with maximum output power of 100 W
(Hewlett-Packard 54600B，USA)． The flow rate of the
process was adjusted to 2 mL /min and controlled by a
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Vol. 25 JIN Sheng-lang，et al:High Intensity Pulsed Electric Fields-Assisted Extraction of
Total Flavonoids from Indocalamus tessellatus Leaves
variable speed pump (with maximum output power of
180 W)． The treatment system consisted of two colline-
ar chambers in series and each one with two stainless
steel electrodes separated by a gap of 0． 15 cm whose
treatment volume was 1． 17 × 10-3 mL (r = 0． 5 mm) ，
the maximum output power of HIPEF applied was 1． 2
kW．
Fig. 1 Processing apparatus of high intensity pulsed e-
lectric field
The pulse number (n)was calculated with the follow-
ing equation:
n = 60πr
2 lf
1000Q (1)
Consequently，the electric field intensity (E)was cal-
culated as:
E =
Vp － p
2l (2)
Where n is the pulse number;f is the frequency (Hz) ;
l is the length of electrode (cm) ;r is the radius of e-
lectrode (cm) ;Q is the flow velocity (mL /s) ;E is the
electric field intensity (kV /cm) ，and Vp-p is the input
voltage shown on oscillograph．
Methods
HIPEF assisted extraction procedure
The I． tessellatus leaves were grounded in a pulverizer
and passed through an 80-mesh sieve，dried to constant
mass in the oven at 60 ℃ for 6 h，cooled and then
stored in a dry cabinet before usage． The extraction
procedure was as follows:
The dried I． tessellatus leaves→ Ground → Weighed→
Fixed quantity → HIPEF extraction (water as the
transmitting media，material-solvent ratio of 1:20)→
Dried → Extracting solution→ Centrifuging→ Ｒemo-
ving ethanol with distillation under reduced pressure
and concentrating → Concentrated solution→ Determi-
nation of total flavonoids content．
Determination of the total flavonoids
Flavonoids content in I． tessellatus leaves was detected
by NaNO2-Al(NO3)3 colorimetry
［7］，at the wavelength
of 510 nm． Taken rutin as the reference material，the
standard curve was established and the regression e-
quation was calculated． After filtration，the filtrate was
diluted into a constant volume． The flavonoids content
in aliquots (1 mL of filtrate)were determined accord-
ing to the NaNO2-Al(NO3)3 colorimetric method，the
absorbency was detected at 510 nm． Based on the re-
gression equation，the total flavonoids content was ob-
tained． And the extraction yield of total flavonoids was
calculated as follows:
Yextract(%)=(M /m)× 100% (3)
Where Yextract is extraction yield of total flavonoids，M is
total flavonoids content in extracting solution (g) ，m is
weight of grounded powder (g)．
Single factor test
Electric field intensity
Total flavonoids extracted from 1 g I． tessellatus leaves
by HIPEF assisted extraction under 65% ethanol，pulse
number of 6，and material-solvent ratio of 1∶ 30． The e-
lectric field intensity was set at 10，20，30，40，50，60
kV /cm．
Pulse number
Total flavonoids extracted from 1 g I． tessellatus leaves
powder by HIPEF assisted extraction under 65% etha-
nol，material-solvent ratio of 1∶ 30 and electric field in-
tensity of 20 kV /cm． The pulse number was set at 0，2，
4，6，8，10 and 12．
Material-solvent ratio
Total flavonoids extracted from 1g I． tessellatus leaves
powder by HIPEF assisted extraction under pulse num-
ber of 8，65% ethanol，and electric field intensity of 20
kV /cm． The material-solvent ratio was set at 1∶ 20，1∶
30，1∶ 40，1∶ 50，1∶ 60 and 1∶ 70．
Concentration of ethanol
Total flavonoids extracted from 1g I． tessellatus leaves
by HIPEF assisted extraction under pulse number of 8，
material-solvent ratio of 1∶ 30 and electric field intensi-
ty of 20 kV /cm． Different ethanol concentrations were
used (35%，45%，55%，65%，75%，85%，and
95%)．
L9orthogonal test (3
4)
8341 Nat Prod Ｒes Dev Vol. 25
According to the single factor test，factors and levels of orthogonal test were designed (Table 1)．
Table 1 Factors and levels of the orthogonal experiment
Level
Factors
Electrical field intensity
A /(kv·cm-1)
Pulse number
B
Ethanol concentration
C /(mL /g)
Material-solvent ratio
D /℃
1 10 6 65% 1∶ 20
2 20 8 75% 1∶ 30
3 30 10 85% 1∶ 40
Ｒesult and Analysis
Standard curve of rutin
Using the concentration of rutin standard solution as
abscissa and the absorbency as y-coordinate，the re-
gression equation was:
Y = 0． 2573X － 0． 0382 (Ｒ2 = 0． 9969) (4)
Where y is absorbance value of sample，x is sample
concentration，Ｒ2 is correlation coefficient．
Single factor tests of HIPEF assisted extraction
Electric field intensity
The total extraction yield of flavonoids was affected by
electric field intensity (Fig． 2)． As the electric field in-
tensity increased from 0 to 20 kV /cm，total extraction
yield of flavonoids from I． tessellatus leaves increased o-
ver time，indicating electric field intensity was critical
for improving the extraction yield of flavonoids． With
the electric field intensity increasing，from the micro
perspective，the potential difference between the inside
and outside of the cell membrane became larger，and
the electroporation of cell membrane occurred． Higher
electric field intensity caused more solvent to enter the
cell membrane and permeated cell membrane． Thus in-
creasing the electric field intensity can increase the ex-
traction yield． However，when the electric field intensity
increased from 20 to 60 kV /cm，the extraction yield of
Fig. 2 Effect of electric field intensity on the extraction
ratio of total flavonoids
flavonoids decreased． The reason might be that some of
flavonoids were decomposed in higher intensity electric
field． Therefore，electric field intensity of 20 kV /cm
was selected for the following experiments．
Pulse number
Fig． 3 showed the effect of HIPEF pulse number on the
extraction yield of flavonoids，no significant difference
of extraction yield of flavonoids was observed when
pulse number exceeded 8． It was reported that external
electric fields (within 20 μs-10 ms and under HIPEF
strength of 1-100 kV /cm) can induce formation of
pores in membranes，and the structure of cell mem-
brane changed ［10］． As the equilibrium potential be-
tween the inside and outside of the cell can be a-
chieved in 20 μs (pulse number = 10) ，increasing
pulse number did not improve extraction yield of fla-
vonoids． Therefore，pulse number of 8 was selected for
the following experiments．
Fig. 3 Effect of pulse number on the extraction ratio of
total flavonoids
Material-to-solvent ratio
Extraction efficiency of total flavonoids from I． tessel-
latus leaves improved as the material-solvent ratio in-
creased (Fig． 4)． The reason might be that when sol-
vent volume increased，extraction efficiency progres-
sively increased because of diffusion of total flavonoids
9341
Vol. 25 JIN Sheng-lang，et al:High Intensity Pulsed Electric Fields-Assisted Extraction of
Total Flavonoids from Indocalamus tessellatus Leaves
in solvent． However，when the material-solvent ratio ex-
ceeded 1∶ 30，the extraction yield did not change signif-
icantly． The reasons might be，at lower material-solvent
ratio，that the concentration of flavonoids in the solution
would be low，and the flavonoids exchange between sol-
id and solvent had reached equilibrium，thus material-
solvent ratio of 1∶ 30 g /mL was selected for the follow-
ing experiments．
Fig. 4 Effect of material-solvent ratio on the extraction
ratio of total flavonoids
Ethanol concentration
The extraction yield of flavonoids from I． tessellatus
leaves was greatly affected by the ethanol concentration
(Fig． 5)． When the ethanol concentration was lower
than 75%，the extraction yield increased with increas-
ing of ethanol concentration，the reason might be that
the solubility of total flavonoids in ethanol solution in-
creased with the increasing of concentration of ethanol．
When the ethanol was higher than 75%，the extraction
yield decreased obviously with the increasing of ethanol
concentration from 80% -95% (v /v) ，the higher con-
centration of ethanol may affect the formation of pores
on the cell membrane due to decreasing of ionic
strength，thus the process of permeating the cell mem-
brane slowed down，and the extraction yield of total fla-
vonoids decreased． Furthermore，higher concentration of
ethanol can cause the protein to coagulate and fla-
vonoids cannot be easily dissolved in lower concentra-
tion of ethanol，so 75% (v /v)ethanol was the optimal
extraction solvent．
Fig. 5 Effect of ethanol concentration on the extraction
ratio of total flavonoids
Optimization of HIPEF assisted extraction
The powdered samples of I． tessellatus leaves were trea-
ted to extract flavonoids by HIPEF． Based on the sin-
gle-factor tests，four variables at three levels (34，Table
1)were adopted for L9 orthogonal test (Table 2) ，the
ANOVA results was listed (Table 3)． Table 2 showed
the effects of four factors on the extraction yield of total
flavonoids from I． tessellatus leaves were as follows:ma-
terial-solvent ratio (D)＞ electrical field intensity (A)
＞ pulse number (B)＞ ethanol concentration (C)． The
optimal combination was D3A2B2C3，namely，material-
solvent ratio of 1∶ 40，electric field intensity of 20 kV /
cm，pulse number of 8 and ethanol concentration of
75% ． This was consistent with the results of single fac-
tor test，indicating that the optimized extraction condi-
tions were reliable． The verification experiment showed
that under the optimal conditions，the extraction yield of
total flavonoids from I． tessellatus leaves can reach
4. 89% ．
Table 2 Ｒesults of orthogonal experiment
No．
Factors
Electrical field intensity
A /
(kV·cm-1)
Pulse number
B
Ethanol
concentration
C /(mL /g)
Material-solvent
ratio
D /℃
Yield /%
1 1 1 1 1 3． 433
2 1 2 2 2 4． 057
3 1 3 3 3 4． 347
4 2 1 2 3 4． 699
5 2 2 3 1 4． 078
0441 Nat Prod Ｒes Dev Vol. 25
6 2 3 1 2 4． 356
7 3 1 3 2 4． 110
8 3 2 1 3 4． 546
9 3 3 2 1 3． 565
yj1 3． 946 4． 081 4． 112 3． 692
y = 4． 132
fe = 2
yj2 4． 378 4． 227 4． 107 4． 174
yj3 4． 074 4． 089 4． 178 4． 531
Δj 0． 432 0． 146 0． 071 0． 839
Primary and secondary factors D ＞ A ＞ B ＞ C
Optimization levels D3A2 B2C3
Table 3 ANOVA analysis results
Variance
Source
Sum of
squares of
deviations
Degree of
freedom Variance F P
A 0． 295 2 0． 1475 29． 50 ＜ 0． 05
B 0． 040 2 0． 02 4． 00
C 0． 010 2 0． 005 1． 00
D 1． 063 2 0． 5315 106． 30 ＜ 0． 05
Error 0． 295 2 0． 1475
Comparison of HIPEF and conventional extraction
method
The yield of HIPEF assisted extraction was higher than
conventional extraction method． Under the same experi-
mental conditions (75% ethanol，material-solvent ra-
tion of 1∶ 40)as well as the same filtering，concentra-
tion and determination procedures，the extraction yield
of flavonoids was 1． 56%，and the microwave assisted
extraction yield of flavonoids was 1． 92% ［3］．
Conclusion
The optimal conditions for HIPEF-assisted extraction of
total flavonoids from I． tessellatus leaves was material-
solvent ratio of 1∶ 40，electric field intensity of 20 kV /
cm，pulse number of 8 and ethanol concentration of
75% ． Under these optimal conditions，extraction yield
of total flavonoids from I． tessellatus leaves can reach
4. 89%，which was 2． 5 times of conventional extraction
method． HIPEF had greatly improved the extraction
yield of flavonoids from I． tessellatus leaves． Two possi-
ble reasons were summed:1)most ions in extract solu-
tion moved fast under HIPEF，the frequency of the mol-
ecules colliding increased，which might promote strong
chemical reaction． 2)After treated with HIPEF with e-
lectric pulse，the narrow pulse would excite material to
self-frequency oscillation，i． e． resonance vibration．
Therefore，materials would produce huge energy by
themselves and accelerate chemical reaction． Extracting
the active materials from natural products by HIPEF-
assisted extraction technology had the advantages of
high extraction efficiency，strong selectivity，good re-
peatability，little time consuming，few solvent，low ener-
gy consumption，environmental benefits． Therefore，
HIPEF-assisted extraction is expected to offer a new
way for the production and analyses of the plant extrac-
tions，and the modernization of pharmaceutical engi-
neering．
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(下转第 1410 页)
1441
Vol. 25 JIN Sheng-lang，et al:High Intensity Pulsed Electric Fields-Assisted Extraction of
Total Flavonoids from Indocalamus tessellatus Leaves
论依据，增加资源的附加值，促进加工企业的发展和
渔业资源的可持续利用，使其发挥更大的社会效益
和经济效益。
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