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: qinsmmu@126.com

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

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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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Table 4 Minimal inhibitory concentration (MIC) and minimal bacterial concentration (MBC) of the aerial part and the rhizome of A.macrocephala

Bacterial species Solvents (mg/ml)

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