全 文 ：Ｒeceived:January 20，2014 Accepted:May 7，2014
Foundation item:Major Progress of National New Drug Ｒesearch
(2012ZX09103201-038)
* Corresponding author Tel:86-21-34204806;E-mail:xbli@ sjtu． edu． cn
天然产物研究与开发 Nat Prod Ｒes Dev 2015，27:126-133
文章编号:1001-6880(2015)1-0126-08
土木香内酯与异土木香内酯的提取与纯化工艺
张 乐，王梦月，李晓波*
上海交通大学药学院中药基因组学实验室，上海 200240
摘 要:本文报道了一种从土木香药材中提取以及纯化倍半萜内酯的方法。乙醇回流提取的方法采用正交试
验设计方法［L9(3
4) ］，考察了 4 个因素对产率与内酯浓度的影响，最终确定了乙醇回流提取的最佳方法:药材
加 10 倍量的 95%乙醇，加热回流提取 1 次，2 h。而硅胶柱层析的最佳纯化方法为:以 100 ～ 200 目的硅胶，1∶ 3
的上样量，径高比为 1∶ 2。其洗脱剂成分为石油醚∶丙酮(100∶ 3，v /v) ，洗脱流速为 35 mL /min。采用该方法，异
土木香内酯与土木香内酯的提取率大于 70%，其纯度大于 90%。
关键词:土木香内酯;异土木香内酯;正交试验;硅胶柱层析;土木香
中图分类号:Ｒ932 文献标识码:A DOI:10． 16333 / j． 1001-6880． 2015． 01． 026
Extraction and Purification of Alantolactone and
Isoalantolcatone from Ｒoot of Inula helenium
ZHANG Le，WANG Meng-yue，LI Xiao-bo*
School of Pharmacy，Shanghai Jiao Tong University，Shanghai 200240，China
Abstract:The extraction and purification method of sesquiterpene lactones from the root of Inula helenium L． was opti-
mized． The tests of orthogonal array design［L9(3
4) ］were performed to analyze the effect of single factors，namely etha-
nol concentration，extraction duration and times of extraction，on the extraction yield of isoalantolcatone and alantolac-
tone． As the result，the optimal extraction conditions were found as follows:the dried roots of I． helenium were extracted
by reflux with 95% ethanol in solid / liquid ratio of 1∶ 10 (w /v)for 2 hrs． The optimized purification process of ethanol
extract was achieved by a pressurized silica gel (100-200 mesh)column chromatography with diameter to height ratio of
1∶ 2． The ratio of mixed sample to silica gel was 1∶ 3 (w /w) ，the eluent composed of petroleum ether (30-60 ℃)and
acetone (100∶ 3，v /v) ，flowed at the rate of 35 mL /min． The extraction yield of the lactones under the optimal method
was above 70%，and the purity was above 90% ．
Key words:Isoalantolcatone;alantolactone;orthogonal array design;silica gel column chromatography;Inula helenium L．
Introduction
Inula helenium L． (Compositae)is a perennial plant，
distributed in many areas of China，Europe and North
America． The root of I． helenium is rich in eudesmane-
type sesquiterpene lactones，such as isoalantolcatone
and alantolactone［1］． In recent years，extensive investi-
gations on active constituents of I． helenium roots
proved that sesquiterpene lactones were responsible for
various biological activities，such as antibacterial，anti-
fungal，hepatoprotective and antitumor activities［2-4］．
Therefore，it is necessary to find an efficient method for
extraction and purification of sesquiterpene lactones．
The current study was mainly focused on the ultrasonic-
assisted extraction of phenolic and microwave-assisted
extraction of flavones［5，6］，but the purity was not repor-
ted． In this study，we report an improved extraction
method for sesquiterpene lactones by ethanol-reflux and
purification by silica gel column chromatography． The
extraction yield of the lactones obtained by the optimal
method was above 70%，and the purity was above
90% ．
Materials and methods
Plant material and reagents
Dried roots of I． helenium were purchased from the
herbal market in Sichuan Province，China． The plant
material was ground and kept in a cool and dark place．
Alantolactone and isoalantolcatone standards were in-
ternally isolated and characterized by spectral methods
(IＲ，NMＲ and MS)［7，8］． The purity of each reference
substance was above 98% as determined by UPLC． All
the solvents used in the experiments were of analytical
grade．
Instruments
UPLC (Agilent 1290) ，Ultraviolet detector (G4212-
60008) ，Ｒotovapor，Electric heating blast drying cham-
ber，Electric balance，etc．
Standard preparation
Each reference substance was dissolved in methanol to
the concentration of 0． 115，0． 230，0． 345，0． 460，
0. 575 and 0． 690 mg /mL of isoalantolactone，and
0. 114，0． 228，0． 342，0． 456，0． 570，and 0． 684 mg /
mL of alantolactone． These samples of reference sub-
stance were injected in triplicate (injection volume 1
μL)． The retention times (ＲTs) of isoalantolactone
and alantolactone were 2． 864 min and 3． 039 min，re-
spectively． Linear regression equations of isoalantolac-
tone and alantolactone were y = 3736． 8x + 21． 674
(Ｒ2 = 0． 9997)and y = 4176x + 25． 284 (Ｒ2 = 0．
9996) ，respectively．
Sample preparation
The sample extracts of I． helenium roots were dissolved
in methanol for UPLC analysis． 1 μL solution of each
sample extract was injected into UPLC system． Contents
of isoalantolactone and alantolactone in the examined
samples were calculated by linear regression equations
and expressed as milligram per milliliter solutions
(mg /mL)．
Each experiment was performed in triplicate． All the
data values were expressed as means with standard de-
viation．
Quantitative determination of isoalantolactone and
alantolactone
The determination of isoalantolactone and alantolactone
content was carried out on an Agilent Zorbax Eclipse
Plus C18 column (2． 1 × 50 mm，1． 8 μm)with a mo-
bile phase consisting of 40% acetonitrile and 60% wa-
ter，at a flow rate of 0． 6 mL /min，and the detection
wavelength was set at 210 nm． The column oven tem-
perature was set at 35 ℃ ． The injection volume was 1
μL． Quantitative data were obtained from the peak are-
as of each sample using an external standard method．
Ethanol-reflux extraction
To optimize the ethanol-reflux extraction conditions，4
parameters were selected and investigated，namely:ratio
of solid to liquid (A) ，solvent concentration (B) ，ex-
traction duration (C)and times of extraction (D)． The
extraction process was further optimized using the or-
thogonal array design shown in Table 1． Dried roots of
I． helenium(20 g)and ethanol were put into flask for
ethanol-reflux extraction with designed ratio of plant
material to ethanol (solid / liquid，w /v) and various
conditions of experiments． The extract was filtered，and
solvent was removed under vacuum． The obtained etha-
nol extract was dried and concentrated under vacuum．
All experiments were performed in duplicate．
The yield (X1)of extracts were calculated based on
the weight of obtained extract and plant material，while
the extraction yield (X3)was defined as the ratio of
the weight of lactones in extracts to the total weight of
lactones in the plant material． The weight of lactones in
extracts was calculated based on the purity (X2)of the
extracts，the ratio of the weight of lactones to the ex-
tracts．
The method of weighed score (Y)comparison was used
to evaluate the effects of every factor on the yield and
purity，and Y was calculated using the following expres-
sion:Y = 0． 8X2 /6． 55-0． 2X1 /66． 37，where 6． 55 was
the average purity of the extracts in group 9 (Table
2) ，which was the best in purity (X2)of the extracts，
while the 66． 37 was the average of the yield (X1)of
the group 4，which was the best in yield． A single factor
analysis of variance (ANOVA)was adopted to investi-
gate the effect of each factor in the process of extrac-
tion．
Purification by silica gel column chromatography
The extracts obtained from the I． helenium roots by an
optimized ethanol-reflux process were further purified
by silica gel column chromatography． The chromato-
graphic column was packed by dry column-packing．
The mixed sample was composed of the ethanol-extracts
721Vol. 27 ZHANG Le，et al:Extraction and Purification of Alantolactone and Isoalantolcatone from Ｒoot of Inula helenium
Table 1 Levels and factors of orthogonal design
Levels
Factors
(A)Ｒatio of solid
to liquid (g /mL)
(B)EtOH
concentration (%)
(C)Extraction
duration (h)
(D)Extraction
times(n)
1 8 65 1 1
2 10 75 2 2
3 12 95 2． 5 3
and chromatographic silica gel (1∶ 1． 5，w /w) ，and the
following factors were investigated in the process of
purification:granularity of chromatographic silica gel，
loading amount，ratio of diameter to height，ingredients
of eluent and flow rate． The purified ethanol-extract was
dried，concentrated under vacuum and further dissolved
in methanol for UPLC analysis．
The method of weighted score (Y) comparison was
used to evaluate every effect of factors on the yield and
purity as well，according to extraction yield of lactones
(X3) ，purity (X2)and the amount of eluent (X4) :Y
=0． 2X3 + 0． 4X2-0． 4X4，where the amount of eluent
(X4) (mL /g)was defined as the ratio of the amount
of eluent used for purifying ethanol extracts to the
weight of plant material for producing the ethanol ex-
tract． Each experiment was performed in triplicate． All
the data values were expressed as means with standard
deviation．
The experimental designs of every factor were as fol-
lows:
Granularity of chromatographic silica gel
Three types (60-100 mesh，100-200 mesh and 200-300
mesh) of granularities of chromatographic silica gel
were tested． The ratio of diameter to height of column
chromatography was 1∶ 5，and the ratio of mixed sample
to silica gel was 1 ∶ 3 (w /w)． The eluent of column
chromatography was composed of petroleum ether (30-
60 ℃)and acetone (100∶ 3，v /v) ，at a flow rate of 5．
5 mL /min．
Loading quantity
The experiment was performed using silica gel (100-
200 mesh)column chromatography by eluent composed
of petroleum ether (30-60 ℃)and acetone (100∶ 3，v /
v) ，at a flow rate of 5． 5 mL /min． Different loading
quantities of mixed sample were tested． The ratios of
mixed sample to silica gel were 1∶ 6，1∶ 4，1∶ 3，1∶ 2，1∶
1 (w /w) ，respectively．
Ｒatio of diameter to height
The experiment was performed by silica gel (100-200
mesh)column chromatography． Different ratios of di-
ameter to height were studied∶ 1∶ 0． 5，1∶ 2，1∶ 5，1∶ 16．
The ratio of mixed sample to silica gel was 1∶ 3 (w /w)
and the eluent was composed of petroleum ether (30-
60 ℃)and acetone 100∶ 3 (v /v)． The eluent flowed at
the rate of 5． 5 mL /min．
Ingredient of eluent
The experiment was performed by silica gel (100-200
mesh) column chromatography whose diameter to
height was 1∶ 5． The ratio of mixed sample to silica gel
was 1 ∶ 3 (w /w)． Different ingredients of eluent were
studied∶ petroleum ether (30-60 ℃)/ acetone 100 ∶ 2，
100∶ 3，100∶ 4，100∶ 5 (v /v)． The eluent flowed at the
rate of 5． 5 mL /min．
Flow rate
The experiment was performed by silica gel (100-200
mesh)column chromatography whose ratio of diameter
to height was 1∶ 5． The ratio of mixed sample to silica
gel was 1 ∶ 3 (w /w)and the eluent was composed of
petroleum ether (30-60 ℃)and acetone 100 ∶ 3 (v /
v)． Three different flow rates were studied∶ 5． 5，9． 0，
35 mL /min． The low flow rate between 5． 0 and 9． 0
mL /min was adjusted by the switch on the column
chromatography，while the high flow rate of 35 mL /min
was achieved by pressurized column chromatography．
Each experiment was performed in triplicate． All the
data values were expressed as means with standard de-
viation．
Ｒesults and Discussion
Extraction by ethanol-reflux
821 Nat Prod Ｒes Dev Vol. 27
Orthogonal array design
The results of each experimental group，yield and purity
of extract，and data analysis were shown in Table 2． Ac-
cording to the Ｒ value，the ethanol concentration was
found to be the most important factor，the order of the
effects of all factors was B ＞ D ＞ A ＞ C． This was also
demonstrated by F and P value of ANOVA (Table 3)．
The ethanol concentration (factor B)and reflux times
(factor D)were markedly correlative with yield of
isoalantolactone and alantolactone． The optimal method
of ethanol-reflux extraction of sesquiterpene lactones
was determined as∶ the ratio of solid to liquid was 1∶ 10
(w /v，g /mL)with 95% ethanol by reflux extraction
for 2 hours，once (A2B3C2D1)．
Table 2 Experimental results and data analysis
Factors Ｒesults (n = 2) Yin(n = 1，2)
No． A B C D
Yield (%)
X11
Purity (%)
X21
Yield(%)
X12
Purity (%)
X22
Yi1 Yi2 Yi =∑Yij
1 1 1 1 1 47． 23 3． 52 43． 77 3． 44 28． 76 28． 83 57． 59
2 1 2 2 2 53． 02 3． 05 49． 91 2． 96 21． 27 21． 11 42． 39
3 1 3 3 3 33． 73 4． 69 30． 03 4． 51 47． 12 46． 03 93． 15
4 2 1 2 3 69． 11 2． 99 63． 63 2． 75 15． 69 14． 41 30． 11
5 2 2 3 1 41． 87 3． 88 35． 78 3． 66 34． 77 33． 92 68． 69
6 2 3 1 2 25． 33 5． 18 22． 76 5． 00 55． 63 54． 21 109． 84
7 3 1 3 2 69． 65 3． 00 62． 87 2． 93 15． 65 16． 84 32． 49
8 3 2 1 3 44． 98 2． 83 40． 08 2． 72 21． 01 21． 14 42． 15
9 3 3 2 1 20． 03 6． 61 18． 99 6． 49 74． 70 73． 54 148． 24
K1 193． 13 120． 19 209． 58 274． 52 K = 624． 66
K2 208． 64 153． 23 220． 74 184． 73 W =28148． 96
K3 222． 89 351． 24 194． 34 165． 41 P = 21677． 75
k1 32． 19 20． 03 34． 93 45． 75
k2 34． 77 25． 54 36． 79 30． 79
k3 37． 15 58． 54 32． 39 27． 57
Ｒj 4． 96 38． 51 4． 40 18． 18
Uj 21751． 61 26882． 38 21736． 28 22807． 75
Qj 73． 87 5204． 63 58． 53 1130． 00
Note:K =∑Yi，W =∑Yi2，P = 1 /18 × K2，Ｒj = Max (Ki)-Min (Ki) ，Uj = 1 /6∑(Ki2) ，Qj = Ui-P
Table 3 Variance analysis of experiments results (ANOVA)
Source Corrected
model Intercept
Sum of squares
Qj
Degree of freedom
f
Mean
square F value
Significant
level
A 73． 87 5204． 63 36． 94 3． 93
B 5204． 63 2 2602． 32 276． 91 ＊＊
C 58． 53 2 29． 27 3． 11
D 1130． 00 2 565． 00 60． 12 ＊＊
Error 4． 18 9 2． 09
Total 6471． 21 17
F0． 01(2，9)= 8． 02;F0． 05(2，9)= 4． 26
Note:QT = W-P，Qe = QT-QA-QB-QC-QD，Fj =(Qj / fj)/(Qe / fe)
921Vol. 27 ZHANG Le，et al:Extraction and Purification of Alantolactone and Isoalantolcatone from Ｒoot of Inula helenium
Verification experiment
Toverify the above the results，three experiments of eth-
anol-reflux extraction were performed using the optimal
method:ratio of solid to liquid was 1 ∶ 10 (w /w，g /
mL) ;ethanol concentration was 95%;extraction time
was 2 hours，once． The result was shown in Table 4．
The extraction yields of 3 groups were above 75% and
their purities were around 7% ． As a result，the opti-
mized method was stable and reliable．
Table 4 Ｒesults of verification experiment
No． Plant material (g) Yield (%)
Content (%)
Isoalantolactone Alantolactone Total lactones
Extraction yield (%)
1 653． 0 24． 86 4． 79 2． 37 7． 16 75． 16
2 650． 0 24． 10 4． 48 2． 69 8． 17 82． 38
3 650． 8 24． 21 4． 94 2． 20 7． 94 81． 09
Purification by silica gel column chromatography
Effects of granularity of chromatographic silica gel
Three types of granularities of chromatographic silica
gel 60-100 mesh，100-200 mesh and 200-300 mesh
were tested for comparing their effects on extraction
yield (X3) ，purity (X2)and amount of eluent (X4) ，
and the results were showed in Fig 1． The experimental
results showed that the increasing of granularity of
chromatographic silica gel would result in lower extrac-
tion yield，in a little higher purity，and remarkably
higher amount of eluent． According to the comparative
equation (Y = 0． 2X3 + 0． 4X2-0． 4X4)． The optimal
granularity of chromatographic silica gel was 100-200
mesh (Fig． 1)．
Fig. 1 Effect of granularity of chromatographic silica gel on extraction yield (a) ，purity (b) ，amount of eluent (c)and com-
prehensive score(d)of isoalantolactone and alantolactone(n = 3)
Effects of loading quantity
A granularity of chromatographic silica gel 100-200
mesh was used to test the effect of loading quantity，by
eluent composed of petroleum ether (30-60 ℃)and
acetone (100∶ 3，v /v)at a flow rate of 5． 5 mL /min．
The effects of different loading quantities of mixed sam-
ple on the extraction yield(X3) ，purity (X2)and a-
mount of eluent (X4)were shown in Fig． 2． The results
demonstrated that with the increasing of loading quanti-
ty，the extraction yield would get lower． In addition，the
increasing of loading quantity would result in a little
higher purity，and remarkably higher amount of eluent．
According to the comparative equation (Y = 0. 2X3 +
0． 4X2-0． 4X4) ，the optimal method was achieved when
the ratio of mixed sample to silica gel was 1∶ 3 (w /w，
g /mL)．
031 Nat Prod Ｒes Dev Vol. 27
Fig. 2 Effect of loading quantity on extraction yield (a) ，purity (b) ，amount of eluent (c) ，and comprehensive score (d)of
isoalantolactone and alantolactone(n = 3)
Effects of the ratio of diameter to height
Based on the results of 2． 1 and 2． 2，different ratios of
diameter to height (1∶ 0． 5，1∶ 2，1∶ 5 and 1∶ 16)were
tested． According to the results，the increasing of the
ratio of diameter to height would result in a little lower
extraction yield，and higher ，purity of lactones． Howev-
er，the amount of eluent was remarkably increased with
the increasing of the ratio of diameter to height (Fig．
3)． Judged by the comparative equation (Y = 0． 2X3 +
0． 4X2-0． 4X4) ，the optimal ratio of diameter to height
was 1∶ 2．
Fig. 3 Effect of the ratio of diameter to height on extraction yield (a) ，purity (b) ，amount of eluent (c) ，and comprehensive
score (d)of isoalantolactone and alantolactone(n = 3)
Effects of ingredient of eluent
As shown in Fig 4，the results indicated that increasing
acetone would result in higher extraction yield，lower ，
purity of lactones，and significantly less amount of elu-
ent． Therefore，the optimal method was achieved when
the eluent was composed of petroleum ether (30-60
℃)and acetone (100 /3，v /v)according to the com-
parative equation (Y = 0． 2X3 + 0． 4X2-0． 4X4)．
Effects of flow rate
Three different flow rates (5． 5，9． 0，35 mL /min)were
tested using silica gel (100-200 mesh)column chro-
matography with ratio of diameter to height 1∶ 5，ratio of
mixed sample to silica gel 1∶ 3 (w /w)and the eluent
composed of petroleum ether (30-60 ℃)and acetone
131Vol. 27 ZHANG Le，et al:Extraction and Purification of Alantolactone and Isoalantolcatone from Ｒoot of Inula helenium
Fig. 4 Effect of ingredient of eluent on extraction yield (a) ，purity (b) ，amount of eluent (c)and comprehensive score(d)
of isoalantolactone and alantolactone(n = 3)
100∶ 3 (v /v)． The results demonstrated that the pres-
surized column chromatography did not have significant
effects on the extraction yield，，purity of lactones，and
the amount of eluent (Fig． 5)． However，it shortened
the time of experiment significantly． As a result，the
pressurized column chromatography can be adopted in
the manufacturing process．
Fig. 5 Effect of flow rate on extraction yield (a) ，purity (b) ，amount of eluent (c)and purification duration (d) (n = 3)
Verification experiment
To verify the results of single factor experiments，3 ex-
periments were performed according to the optimal con-
dition∶ the ratio of mixed sample to silica gel was 1 ∶ 3
(w /w) ;granularity of chromatographic silica gel was
100-200 mesh;ingredient of eluent was petroleum ∶ e-
ther (30-60 ℃)/ acetone (100 ∶ 3，v /v) ;the ratio of
diameter to height was 1 ∶ 2，at a flow rate of 35 mL /
min． As shown in Table 5，the extraction rates (X3)of
the obtained lactones among the 3 groups were above
85%，and their purities were above 90%． As a result，the
optimized method of purification was stable and reliable．
Table 5 Ｒesults of verification experiments of purification process
No． Mixed sample (g) Yield (%)
purity (%)
Isoalantolactone Alantolactone Total lactones
Amount of
eluent (mL /g)
Extraction
yield (%)
1 15． 097 6． 53 73． 65 17． 94 91． 59 14． 2 85． 81
2 15． 127 6． 67 72． 70 19． 07 91． 77 15． 1 87． 82
3 15． 063 6． 61 73． 29 17． 83 91． 13 15． 8 86． 42
231 Nat Prod Ｒes Dev Vol. 27
Conclusion
The results showed that the method of ethanol-reflux
extraction and purification by silica gel column chroma-
tography was stable and reliable for producing isoalan-
tolactone and alantolactone from I． helenium roots． The
optimal ethanol-reflux extraction was achieved when the
plant material was extracted by reflux with 95% etha-
nol in solid / liquid ratio of 1∶ 10 (w /v)for 2 hrs． The
optimal purification method was performed by pressur-
ized silica gel (100-200 mesh)column chromatography
whose ratio of diameter to height was 1∶ 2． The ratio of
mixed sample (ethanol extract / silica gel，1∶ 1． 5，w /w)
to silica gel was 1∶ 3 (w /w) ，and the eluent was com-
posed of petroleum ether (30-60 ℃) and acetone
(100∶ 3，v /v)which flowed at the rate of 35 mL /min．
By this producing method，the extraction yield of ses-
quiterpene lactones from I． helenium roots was above
70%，and their purities were above 90% ．
Acknowledgments
The authors acknowledge the financial support from the
project 2012ZX09103201-038 by the Major progress of
National new drug research．
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