comparative research of chemical constituents and bioactivities between petroleum ether extracts of...
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ORIGINAL RESEARCH
Comparative research of chemical constituents and bioactivitiesbetween petroleum ether extracts of the aerial partand the rhizome of Atractylodes macrocephala
Wei Peng • Ting Han • Wen-Bo Xin • Xiao-Gang Zhang •
Qiao-Yan Zhang • Min Jia • Lu-Ping Qin
Received: 26 July 2009 / Accepted: 13 January 2010 / Published online: 29 January 2010
� Springer Science+Business Media, LLC 2010
Abstract A new possible resource of antimicrobial and
antineoplastic was discovered for the first time from the
aerial part of Atractylodes macrocephala Koidz. which was
commonly disposed as waste. A comparative analysis of
the constituents from the petroleum ether extracts of the
aerial part and the rhizome of A. macrocephala was
investigated by gas chromatography–mass spectrometry
(GC/MS). Total of 21 compounds of the aerial part and 31
compounds of the rhizome of A. macrocephala were
determined. Although the extracts of the aerial part and the
rhizome showed little chemical composition correlation,
both of them demonstrated antimicrobial and antitumor
activities. Furthermore, the aerial part showed better
cytotoxic activities than the rhizome with CEM cell line of
IC50 values being below 10 lg/ml. These results could
indicate that fatty oils from the aerial part of A. macro-
cephala had great potential to be used as a source for
natural medicines or health products.
Keywords Atractylodes macrocephala Koidz. �GC/MS analysis � Antimicrobial � Antitumor
Introduction
Atractylodes macrocephala Koidz., belonging to the
Compositae family, is widely distributed in China. The
rhizomes of A. macrocephala Koidz. have been used as an
important crude drug against stomach diseases, digestive
disorders, anorexia, and were listed in Shen-nong-ben-cao-
jing, the first Chinese pharmacopoeia written in the Han
dynasty. Recently, the rhizomes of A. macrocephala have
been also reported to show a variety of other pharmaco-
logical activities, such as antitumor (Zhang et al., 2006),
anti-inflammatory (Endo and Taguchi, 1979; Kwan et al.,
1989) and antioxidant (Kwan et al., 1989). Previous phy-
tochemical investigations into A. macrocephala showed the
presence of polyacetylenes (Chen, 1989), sesquiterpenoids
(Chen et al., 1997; Huang et al., 1992; Zhang et al., 1998)
and sesquiterpene glycosides (Kitajima et al., 2003). In
China, it is estimated that annually about 7,000 tonnes of
the rhizome of A. macrocephala are demanded. However,
most previous study about A. macrocephala focused on the
chemical constituents and pharmacological effects of the
rhizomes but no reports about the aerial part of A. mac-
rocephala because of the traditional custom use of rhizome
of this plant. The aerial part of A. macrocephala weighs
approximately 60% of the total mass of the plant, but most
of them are commonly disposed in landfills without any
pretreatment because it was traditionally useless. There-
fore, development of the aerial part as value-added prod-
ucts becomes a new pathway for full use of the resource of
A. macrocephala.
To the best of our knowledge, there have been no reports
on the aerial part of A. macrocephala extracts with
chemical components or pharmacological activities. In this
study, we first compared the chemical components and
pharmacological activities between the rhizome and the
aerial part of A. macrocephala. We also provided the
first evidence of antimicrobial and cytotoxic properties
of the lipid soluble components from the aerial part of
A. macrocephala, which will provide the possibility for
Wei Peng and Ting Han equally contributed to this manuscript; both
should be considered as first author.
W. Peng � T. Han � W.-B. Xin � X.-G. Zhang � Q.-Y. Zhang �M. Jia � L.-P. Qin (&)
School of Pharmacy, Second Military Medical University,
325 Guohe Rd, Shanghai 200433, China
e-mail: [email protected]
123
Med Chem Res (2011) 20:146–151
DOI 10.1007/s00044-010-9311-8
MEDICINALCHEMISTRYRESEARCH
application of the aerial part as a disease preventive or
therapeutic agent.
Methods
Materials
The whole A. macrocephala plant was collected from Pan
An, Zhejiang province, P.R. China in September 2008, and
identified by Professor L.P. Qin. A voucher specimen has
been deposited in School of Pharmacy, Second Military
Medical University (SMMU), Shanghai, China. The plant
material was dried at room temperature and powdered prior
to extraction.
Preparation of the crude extracts and petroleum ether
extracts
The dried and powdered rhizome and the aerial part were
extracted five times (each extraction period lasting 4 days)
with 75% ethanol aqueous solution repeatedly by macera-
tion, respectively. The materials were filtered, and the clear
supernatant was then concentrated under reduced pressure
at 50�C with a vacuum rotary evaporator. A sample of the
concentrated ethanol extracts was partitioned between
water and petroleum ether (60–90�C). After removing the
petroleum ether fraction, the aqueous layer was further
partitioned between acetic ether and n-butanol. The
extracts of petroleum ether, acetic ether, and n-butanol
were evaporated and the residues were used in the fol-
lowing experiments.
Gas chromatography/mass spectrometry (GC/MS)
analysis
Chromatography was performed on a Thermo Focus DSQ
gas chromatograph with a mass-selective detector with
electron impact ionization. Analytes were separated using a
HP-5MS capillary column of 30 m 9 0.25 mm with a
phase thickness of 0.25 lm from HP, which was inserted
directly into the ion source of the MS system. The tem-
perature program used for analysis was as follows: the
initial temperature was 100�C for 2 min, which was
increased to 300�C at 15�C/min; 300�C was maintained for
8 min. Helium (99.999%) was the carrier gas maintained at
a flow-rate of 1 ml/min. The split rate was 50:1 and inlet
volume was 1.0 ll. The electron impact ionization condi-
tions were: ion energy 70 eV and the mass range scanned
was 41–450 a.m.u in the full-scan acquisition mode.
Compounds were identified using the NIST Mass Spectral
Search Program (National Institute of Standards and
Technology, Washington, DC, USA). The relative amounts
of individual components of the fatty oils were expressed
as percentages of the peak area relative to the total peak
area.
Test for antibacterial activity
The antibacterial activities of the samples were determined
on a number of bacteria follows: Staphylococcus aureus,
Escherichia coli, Bacillus subtilis and Shigella felxneri
were used.
Determination of the minimal inhibitory concentration
(MIC) and minimal bactericidal concentration (MBC) were
carried out by the broth dilution method (Adesokan et al.
2007). Dilutions of the ether extract were prepared as
follows: 40, 20, 10, and 5 mg/ml. The sterile broth
employed for sample dilution was supplemented with
tween-80 (Merck, Germany) at a concentration of 5% (May
et al., 2000; Hicheri et al., 2003), to enhance ether extract
solubility. MIC values were taken as the lowest ether
extract concentrations that prevent visible bacterial growth
after 24 h of incubation at 37�C, and MBC as the lowest
concentration that completely inhibited bacterial growth.
Each experiment was repeated three times.
Test for antitumor activity
Test cells were grown in RPMI 1640 including 100 units/
ml penicillin and 100 lg/ml streptomycin supplemented
with 15% new-born bovine serum (NBS) at 37�C in a 5%
CO2 atmosphere. For experimentation, the exponentially
growing cells (4–6 9 104) were used. Then, cells were
incubated in the presence of 1,000 lg/ml samples in
DMSO for 72 h at 37�C. After removal of the sample
solution and washing with phosphate-buffered saline (pH
7.4), 10 ll/well of 0.5% 3-(4, 5-dimethyl-2-thiazolyl)-2,
5-diphenyl-2H-tetrazolium bromide cells (MTT) phos-
phate-buffered saline solution was added. After a further
4 h of incubation, 0.04 M HCl was added. Viable cells
were determined by measuring the absorbance at 570 nm.
Measurements were performed three times, and the con-
centration required for a 50% inhibition of viability (IC50)
was determined graphically.
Results and discussion
Chemical components of the aerial part and the rhizome
Color of ether extract of the aerial part was pale green, and
the rhizome was brown. Total of 21 compounds (repre-
senting 90.61%) in the petroleum ether extract of the aerial
part (Table 1) and 31 compounds (representing 99.61%) in
the rhizome (Table 2) were identified. The components
Med Chem Res (2011) 20:146–151 147
123
identified, their retention time and percentage composition
from the aerial part and the rhizome extract are summa-
rized in Tables 1 and 2, respectively. Indeed, the compo-
sition of petroleum ether extract of the aerial part showed
richness in triterpene (70.87%) and fatty acids and fatty
acid esters (14.43%). While the rhizome were character-
ized by the prevalence of sesquiterpene hydrocarbons
(98.29%) (Table 3).
According to our results, the main constituents of the
petroleum ether extract from the aerial part were lupeol
(42.18%), b-amyrin (24.45%), and oleanen-12-en-3-yl ace-
tate (3.67%). The extract of the rhizome obtained by petro-
leum ether extraction consisted mainly of 3, 6-dimethyl-5-
(prop-1-en-2-yl)-6-vinyl-4, 5, 6, 7-tetrahydrobenzofuran
(72.49%) and Guaia-3, 9-diene (7.12%). It is apparent from
the data that the components of the aerial part was predom-
inantly triterpenoids, such as lupeol (42.18%) and b-amyrin
(24.45%), which were previously reported as cancer inhibi-
tor (Keishi and Kyoko, 2000; Rabi and Banerjee, 2009).
However, the rhizome was composed mainly of sesquiter-
penes which have been reported to show attractive activities
against several human tumor cell lines (Itokawa et al., 1985;
Zhang et al., 2002) and bacteria. This study demonstrates
that compositions from the two parts are qualitatively and
quantitatively different.
Antibacterial activity
The antimicrobial activities of the petroleum ether
extracts from the aerial part and the rhizome of A. mac-
rocephala were quantitatively assessed and the results
were presented in Table 4. The petroleum ether extracts
were tested against four human pathogenic bacterias: S.
aureus, E. coli, B. subtilis, and S. felxneri. From Table 4
shown above, the petroleum ether extracts of the aerial
part and the rhizome of A. macrocephala displayed anti-
bacteria activities against the tested organisms. The data
demonstrated that the petroleum ether extracts of the
aerial part of A. macrocephala have broader antimicrobial
spectrum and stronger toxicity to the tested microbes,
especially the MIC about E. coli was 5 mg/ml. This
activity may be attributed to the presence of a high
concentration of triterpene (70.87%) (Nick et al., 1995),
such as lupeol (42.18%).
At present, many antibiotics have encountered drug
resistance or caused severe adverse drug reactions, and
there is an urgent need to search for new antibiotics.
Several recent studies have reported the isolation of novel
compounds with antimicrobial activity from the herbal
medicine. Thus, the aerial part of A. macrocephala might
be used as a source for oral antibacterial drugs.
Table 1 Chemical composition
of the aerial part extract of A.macrocephala
RT retention time
Compound RT Content/%
Ylangene 8.06 0.56
4a-methyl-1-methylene-7-(prop-1-en-2-yl)decahydronaphthalene 8.91 0.54
1-methyl-2,4-di(propan-2-ylidene)-1-vinylcyclohexane 9.26 0.37
3,6-dimethyl-5-(prop-1-en-2-yl)-6-vinyl-4,5,6,7-tetrahydrobenzofuran 10.00 0.21
9-Eicosyne 10.76 0.36
3-Eicosyne 10.89 0.19
3,7,11,15-Tetramethyl-2-hexadecen-1-ol 10.99 0.23
n-Hexadecanoic acid 11.42 3.16
Ethyl palmitate 11.56 2.11
2-hydroxy-5-(3-methylbut-2-enyl)-4-(prop-1-en-2-yl)cyclohepta-2,4,6-trienone 11.95 0.48
Ethyl 1-cyclohexenyl-4-oxobicyclo[3.1.0]hexane-6-carboxylate 11.98 0.32
2,2,8-trimethyl-1,2,3,3a,8,8a-hexahydroazulene-5,6-dicarbaldehyde 12.20 0.36
(9Z,12Z,15Z)-octadeca-9,12,15-trienoic acid 12.32 3.70
(9E,12E)-ethyl octadeca-9,12-dienoate 12.38 2.02
(9Z,12Z,15Z)-ethyl octadeca-9,12,15-trienoate 12.42 1.63
Squalene 15.27 1.03
A-Neooleana-3(5),12-diene 16.38 0.57
b-Sitosterol 20.34 2.47
b-Amyrin 21.10 24.45
Lupeol 21.87 42.18
Oleanen-12-en-3-yl acetate 22.27 3.67
Total identified 90.61
148 Med Chem Res (2011) 20:146–151
123
Table 2 Chemical composition of the rhizome extract of A. macrocephala
Compound RT Content (%)
7,7-dimethyl-1-vinylbicyclo[2.2.1]heptan-2-one 7.88 0.05
Aristolene 7.96 0.03
(1E,5Z)-1,5-dimethyl-8-(prop-1-en-2-yl)cyclodeca-1,5-diene 8.18 0.02
1,1,3a,7-tetramethyl-1a,2,3,3a,4,5,6,7b-octahydro-1H-cyclopropa[a]naphthalene 8.21 0.1
(Z)-4,11,11-trimethyl-8-methylenebicyclo[7.2.0]undec-4-ene 8.33 0.1
1-isopropyl-4,7-dimethyl-1,2,4a,5,8,8a-hexahydronaphthalene 8.36 0.13
1-methyl-2-(prop-1-en-2-yl)-4-(propan-2-ylidene)-1-vinylcyclohexane 8.45 1.10
a-Caryophyllene 8.70 0.33
3,5,5,9-tetramethyl-2,4a,5,6,7,8-hexahydro-1H-benzo[7]annulene 8.74 0.06
1,1,4,7-tetramethyl-1a,2,3,4,4a,5,6,7b-octahydro-1H-cyclopropa[e]azulene 8.80 0.08
2-methyl-5-(6-methylhept-5-en-2-yl)cyclohexa-1,3-diene 8.85 0.08
4a-methyl-1-methylene-7-(prop-1-en-2-yl)decahydronaphthalene 8.92 1.56
Cedrene 9.05 0.29
1,1,3a,7-tetramethyl-1a,2,3,3a,4,5,6,7b-octahydro-1H-cyclopropa[a]naphthalene 9.16 0.26
Guaia-3,9-diene 9.22 7.12
Seychellene 9.26 1.98
1-methyl-2-(prop-1-en-2-yl)-4-(propan-2-ylidene)-1-vinylcyclohexane 9.38 1.19
b-Vatirenene 9.41 0.71
3,6-dimethyl-5-(prop-1-en-2-yl)-6-vinyl-4,5,6,7-tetrahydrobenzofuran 10.02 72.49
4,8a-dimethyl-6-(prop-1-en-2-yl)-3,5,6,7,8,8a-hexahydronaphthalen-2(1H)-one 10.42 1.15
Velleral 11.15 0.66
(E)-4,4-dimethyl-3-(3-methylbut-3-enylidene)-2-methylenebicyclo[4.1.0]heptane 11.33 2.36
n-Hexadecanoic acid 11.42 0.61
2-(4a,8-Dimethyl-2,3,4,4a,5,6-hexahydro-naphthalen-2-yl)-prop-2en-1-ol 11.49 0.39
4,40-Dimethyl-2,20-dimethylenebicyclohexyl-3,30-diene 11.92 0.32
2-hydroxy-5-(3-methylbut-2-enyl)-4-(prop-1-en-2-yl)cyclohepta-2,4,6-trienone 11.95 1.93
2,2,8-trimethyl-1,2,3,3a,8,8a-hexahydroazulene-5,6-dicarbaldehyde 12.15 0.35
Eudesma-5,11(13)-dien-8,12-olide 12.2 1.84
(9Z,12Z)-octadeca-9,12-dienoic acid 12.28 1.04
2-(4a,8-Dimethyl-2,3,4,4a,5,6,7-octahydro-naphthalen-2-yl)-prop-2en-1-ol 12.34 1.05
(9Z,12Z)-ethyl octadeca-9,12-dienoate 12.38 0.23
Total identified 99.61
RT retention time
Table 3 Comparison of
composition of petroleum ether
extract between the aerial part
and the rhizome of
A. macrocephala
Content (%)
The aerial part
of A. macrocephalaThe rhizome of
A. macrocephala
Fatty acids and fatty acids esters 14.43 1.27
Steroids 2.47
Terpenoids 73.71 98.34
Monoterpene hydrocarbons 0.05
Sesquiterpene hydrocarbons 2.84 98.29
Triterpene 70.87
Total 90.61 99.61
Med Chem Res (2011) 20:146–151 149
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Antitumor activity
The investigation on antitumor activity of the petroleum
ether extract from the aerial part and the rhizome of
A. macrocephala was conducted, and the results are rep-
resented in Table 5. From the data shown, the petroleum
ether extract from the rhizome of A. macrocephala dis-
played moderate inhibitory activities against all test tumor
cells when compared with DOX. While the aerial part
possessed stronger cytotoxic activities than the rhizome
with IC50 values on CEM cell line being below 10 lg/ml.
In addition, the aerial part of A. macrocephala also showed
significant cytotoxic effects on MKN-45 cell line and Lovo
cell line.
Cancers are the most life-threatening health problems in
the world (Jemal et al., 2007). Although many different
types of anti-tumor agents are available, severe side effects
and toxicity limit their applications, and there is an urgent
need to search for new antineoplastic agents. Oriental
herbal medicine including traditional and folk-healing
methods has been used for the treatment of cancer for
thousands of years. It means that herbal medicines have
great potential to be developed as natural medicines or
health products for prevention or treatment of cancer. In
this study, we have demonstrated the potential anti-tumor
efficacy of the aerial part of A. macrocephala. However, to
our knowledge, there is no report about cancer inhibitor
prepared from the aerial part of Atractylodes genus plants
in the open literature.
According to the information presented, because of the
diversity and complexity of the natural mixtures of bioac-
tive compounds in the crude plant extract, it is rather
difficult to characterize every compound present and elu-
cidate its structure in a single study. However, further
investigation is still needed to discover the unidentified/
unknown bioactive constituents in the aerial part of
A. macrocephala.
Acknowledgment This research was supported by the Science and
Technology Planning Foundation of Zhejiang province, China (Grant
No. 2008C13035-2).
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Bacterial species Solvents (mg/ml)
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