全 文 ：天然产物研究与开发 Nat Prod Res Dev 2012，24:1070-1074
文章编号:1001-6880(2012)08-1070-05
Received March 14，2011;Accepted June 21，2011
Foundation Item:This project is supported by the Xinjiang Natural Sci-
ence Fund (No． 200821181)
* Corresponding author Tel:86-018911538068;E-mail:jysi@ implad．
ac． cn．
密花香薷挥发油化学成分及其抗菌、抗病毒活性的研究
刘 艺1，斯建勇1* ，曹 丽1，贾晓光2，李晓瑾2
1中国医学科学院 ＆北京协和医学院 药用植物研究所，北京 100193;
2新疆中药民族药研究所，乌鲁木齐 830002
摘 要:采用水蒸气蒸馏法提取密花香薷挥发油，测得密花香薷挥发油的得率为 0． 12%。利用气相色谱 －质谱
(GC-MS)联用技术对密花香薷挥发油的化学成分进行了分析研究，鉴定了 26 种化合物，占挥发油总量的 76．
04%。体外抗菌试验和抗病毒试验的结果表明:密花香薷挥发油具有抗菌和抗病毒作用。
关键词:密花香薷;挥发油;抗细菌活性;抗真菌活性;抗病毒活性;GC-MS
中图分类号:Q946． 91;R284． 2 文献标识码:A
Chemical Composition，Antimicrobial and Antiviral Activities of
the Essential Oil of Elsholtzia densa Benth．
LIU Yi1，SI Jian-yong1* ，CAO Li1，JIA Xiao-guang2，LI Xiao-jin2
1 Institute of Medicinal Plant Development，Chinese Academic of Medical Science，Peking Union Medical College，Beijing 100193，China;
2Xinjiang Institute of Chinese Materia Medica and Ethnical Materia Wulumuqi 830002，China
Abstract:The essential oil of Elsholtzia densa Benth has been obtained by using water distillation and analyzed by GC-
MS． The content of the essential oil of E． densa was 0． 12% ． Retention indices and mass spectral data were used to iden-
tify 26 oil components，represented 76． 04% of the total essential oil． In the antimicrobial and antiviral activities assay，
the essential oil exhibited antimicrobial and antiviral activities．
Key words:Elsholtzia densa Benth．;essential oil;antimicrobial activity;antiviral activity;GC-MS
Introduction
There are about 40 species of Elsholtzia Willd in the
world． More than 50 compounds have been isolated
from Elsholtzia species． The chemical constituents can
be divided into essential oil，flavonoids，lignanoids，cou-
marins，steroids and fatty acids［1］． There are about 100
chemical constituents of the essential oil analyzed by
GC or GC-MS． From the former literature，we found that
the main chemical constituents of the essential oil from
the same Elsholtzia species are not the same． For exam-
ple，Zhang et al． found that the main chemical constituents
of the essential oil of Elsholtzia densa Benth are Germae-
rene D and D-Limonene［2］，but Wang et al． found that 1，
8-p-Menthadiene and Ocimene are the main chemical
constituents of the essential oil of E． densa［3］．
There are many bioactivities of Elsholtzia species，such
as antibacterial，antiviral，analgesic and sedative activi-
ties［4，5］． In folk medicine，the most common uses of
Elsholtzia species are for the treatment of cold，fever，
dysentery，digestion disorder，heat stroke and so on ［1］．
In this paper，we describe the chemical composition，
antimicrobial and antiviral activities of the essential oil
of E． densa to providing evidences for further develop-
ment of E． densa．
Materials and Methods
Plant material
The aerial parts of E． densa were collected from Xinjiang
Province of China． A voucher specimen was deposited at
the herbarium，Department of Chemistry，Institute of Me-
dicinal Plant Development，Chinese Academic of Medical
Science，Peking Union Medical College，China．
Extraction of the essential oil
DOI:10.16333/j.1001-6880.2012.08.030
The essential oil of E． densa was obtained by using water
distillation for 6 h． The resulted essential oil was stored in
sealed glass vials at 4 ℃ prior to further analysis． The
content of the essential oil of E． densa was 0. 12%．
GC-MS analysis
Gas chromatography-mass spectrometry (GC-MS) a-
nalysis was performed on a Varian 450 gas chromato-
graph equipped with Varian 320-MS detector． The col-
umn used was a capillary column Varian VF-5 MS
CP8944(30 m × 0． 25 mm i． d．，film thickness 0． 25
μm)． The instrument was operated under electron-im-
pact (EI)mode at 70 eV．
Helium was the carrier gas with a flow rate of 1． 0 mL /
min． The injected volume was 1 μL． The split ratio was
1∶ 20． The transfer line and injector temperature were
both 250 ℃ ． The oven temperature program was as fol-
lows:60 ℃ initial temperature，hold for 1 min;in-
creased at 5 ℃ /min to 250 ℃，hold for 10 min;in-
creased at 15 ℃ /min to an interface temperature of
280 ℃ ． Identification of oil components was achieved
based on their retention indices (determined with refer-
ence to a homologous series of normal alkanes) ，and by
computerized matching of the acquired mass spectra
with those stored in Wiley /NIST mass spectral library
of the GC /MS data system．
Antimicrobial activity
Microorganisms
Gram-negative bacteria: Pseudomonas aeruginosa
(ATCC10104)and Escherichia coli (ATCC2592);Gram-
positive bacteria:Staphylococcus aureus(ATCC25923);
fungi:Candida albicans(ATCC10231)． All of these mi-
croorganisms were obtained from the pharmacology la-
boratory of Institute of Medicinal Plant Development，
Chinese Academic of Medical Science and Peking U-
nion Medical College．
Antimicrobial activity assay
The antimicrobial activity of the essential oil against
microorganisms was tested by a micro-well dilution
method ［6］． Microorganisms were cultured overnight at
37 ℃ on［Nutrient broth(NB)for bacteria and Sab-
ouraud dextrose broth(SDB)for fungi］and adjusted to
a final density of (105 CFU /mL for bacteria and 103
CFU /mL for fungi) ，and used as an inoculum． The es-
sential oil was first dissolved in DMSO and then diluted
with broth． The initial concentration of DMSO never ex-
ceeded 2． 5% ． Two-fold serial dilutions were made in
broth over a range to give final concentrations of 100 to
1． 56 mg /mL for Gram-negative bacteria，80 to 1． 25
μg /mL for Gram-positive bacteria and 80 to 0． 78 mg /
mL for fungi． The 96-well plates were prepared by dis-
pensing into each well 95 μL of (NB for bacteria and
SDB for fungi)and 5 μL of the inoculum． 100 μL of
the essential oil initially prepared was added into the
first wells． The last well containing 195 μL of (NB for
bacteria and SDB for fungi)without the essential oil
and 5 μL of the inoculum on each strip was used as
negative control． The final volume in each well was 200
μL． The reference test compounds were (penicillin for
bacteria and itraconazole for fungi)． The solvent of es-
sential oil was used as a negative control． The contents
of the wells were mixed and the microplates were incu-
bated at (37 ℃ for bacteria and 35 ℃ for fungi)for
24 h． The minimal inhibitory concentration (MIC)was
defined as the lowest dilution at which no growth was
detected． Each test was repeated thrice．
Antiviral activity
Viruses and cells
Influenza viruses (A /H3N2 /Jifang /15 /90 and B /Jif-
ang /13 /97) and Madin-Darby canine kidney (MD-
CK)cells were obtained from Institute of Medicinal Bi-
otechnology，Chinese Academic of Medical Science and
Peking Union Medical College． The virus was propaga-
ted in chorio-allantoic cavities of 10-day old embryo-
nated hen eggs at 35 ℃ for 3 days． The infected allan-
toic fluids were clarified by centrifugation at 1000 × for
20 min and stored as virus stock solution in small por-
tions at -80 ℃ ．
Antiviral activity assay
The inhibition of virus-induced cytopathic effect
(CPE)was determined by visual (microscopic)exam-
ination of cells． The essential oil was dissolved in DM-
SO and added to Sabouraud medium in order to get a
appropriate initial concentration． In general，eight con-
centrations of the essential oil，each varying by one-half
log10 from the next，were evaluated in MDCK cells．
MDCK cells were seeded onto a 96-well culture plate at
1701
Vol. 24 LIU Yi，et al:Chemical Composition，Antimicrobial and Antiviral
Activities of the Essential Oil of Elsholtzia densa Benth．
a concentration of 10-5 cells per well for influenza A vi-
rus and 10-3 cells per well for influenza B virus． Then
the culture plates were incubated at 37 ℃ in 5% CO2
for 24 h． After 24 h，these cells were infected with in-
fluenza A and B viruses for 2 h． Then medium was re-
moved and the 96-well plates were replaced with media
containing serially diluted essential oil． The culture
plates were incubated at 37 ℃ in 5% CO2 for 36 h un-
til appropriate CPE was achieved． Ribavirin was used
as a positive control，and DMSO was used as a negative
control． CPE inhibition data were expressed as the 50%
effective (viral CPE-inhibitory ) concentration
(EC50) ，50% cytotoxicity (cell-inhibitory)concentra-
tion (CC50)and selectivity index (SI) ，determined as
the CC50 /EC50 ．
Results and Discussion
Chemical composition of the essential oil
Water-distilled essential oil from aerial parts of E． den-
sa were analyzed by GC-MS and resulted in the identi-
fication of 26 compounds representing 76． 04% of the
oil (Table 1)． The dominant compounds are as fol-
lows:α-bisabolol (14． 97%) ，elemene (9． 04%) ，β-
selinene (8． 97%) ，(+)-γ-gurjunene (6． 37%) ，α-
guaiene (5． 92%) ，patchoulene (3． 96%) ，α-ylange-
ne (3． 90%) ，bulnesol (3． 6%) ，and caryophyllene
(2． 15%)． The results are different from the former lit-
erature［2，3］． The reason for the differences may be that
the aerial parts of E． densa used in our study and other
studies were collected from different places．
Table 1 Composition of the essential oil of E． densa
Compound Formula RI Relativecontent (%)
Similarity
(%)
Linalool C10H18O 1101 0． 74 96
(Z)-2，6-Dimethyl-6，8-nonadien-4-one C11H18O 1171 2． 12 92
2，6-Dimethyl-3，7-octadiene-2，6-diol C10H18O2 1199 1． 09 93
Elemene C15H24 1391 9． 04 90
Caryophyllene C15H24 1418 2． 15 97
α-Guaiene C15H24 1442 5． 92 92
(+)-γ-Gurjunene C15H24 1459 6． 37 90
1，8-Dimethyl-4-isopropenylspiro［4． 5］dec-7-ene C15H24 1463 1． 10 93
α-Ylangene C15H24 1471 3． 90 98
Patchoulene C15H24 1474 3． 96 92
(-)-Alloaromadendrene C15H24 1478 1． 24 86
γ-Cadinene C15H24 1488 1． 16 90
Eremophilene C15H24 1502 2． 02 85
β-Selinene C15H24 1508 8． 97 94
13-nor-cis-Eudesm-6-en-11-one C14H22O 1598 1． 18 96
(+)-Calarene C15H24 1603 0． 23 88
1，5，5，8-Tetramethyl-12-oxabicyclo［9． 1． 0］dodeca-3，7-diene C15H24O 1619 0． 41 91
Hinesol C15H26O 1636 1． 66 87
α-Eudesmol C15H26O 1652 0． 54 92
Bulnesol C15H26O 1666 3． 60 96
α-Bisabolol C15H26O 1685 14． 97 97
(E，E)-3，7-Dimethyl-10-(1-methylethylidene)-3，7-cyclodecadien-1-one C15H22O 1693 1． 64 93
Phytone C18H36O 1842 0． 44 88
α-Cadinene C15H24 1718 0． 79 95
2701 Nat Prod Res Dev Vol. 24
(E，Z)-3，7，11-Trimethyl-2，6，10-dodecatrien-1-ol C15H26O 1748 0． 64 91
n-Hexadecanoic acid C16H32O2 1959 0． 16 87
Total 76． 04
Antimicrobial activity
The essential oil of E． densa was found to be inhibiting
both Gram-positive bacteria (S． aureus) and Gram-
negative bacteria (P． aeruginosa and E． coli)． The es-
sential oil exhibited slight activity against P． aeruginosa
and E． coli，but strong activity against S． aureus(Table
2)． The significant antimicrobial activity against S． au-
reus could indicate its potential as a good Antibacterial
agent． It has frequently been reported that Gram (+)
bacteria are more susceptible to essential oil than Gram
(-)bacteria ［7］ and our results did show the selectivity
towards Gram (+)bacteria．
The essential oil exhibited moderate activity against C．
albicans． The minimum inhibitory concentration (MIC)
values were determined by comparison with itraconazole
(MIC = 0． 0503 × 10-3 mg /mL)as a standard agent．
The MIC of the essential oil against C． albicans is 2． 5
mg /mL． The blind control (DMSO)did not inhibit any
of the microorganisms tested．
Table 1 Antibacterial activity of the essential oil of Elsholt-
zia densa Benth
Bacteria
MIC (mg /mL)
Essential oil Penicillin
S． aureus 0． 005 0． 125 × 10-3
P． aeruginosa 25 2． 5
E． coli 50 0． 01
Antiviral activity
The essential oil of E． densa was investigated for its an-
tiviral activity against two influenza viruses (A /H3N2 /
Jifang /15 /90 and B /Jifang /13 /97)． MDCK is sensi-
tive to influenza virus［8］． The results of anti-influenza
virus efficacy in MDCK cells by CPE methods are listed
in (Table 3)． The essential oil exhibited slight activity
against influenza A virus compared with the positive
controls，ribavirin． However，the essential oil did not
show any antiviral activity against influenza B virus
(Table 3)．
Table 1 The anti-influenza virus activity of complexes in MDCK cells
Complex CC50(μg /mL)a
A /H3N2 /Jifang /15 /90 B /Jifang /13 /97
EC50(μg /mL)b，c SId EC50(μg /mL) SId
Essential oil 513． 2 229． 9 2． 23 ＞ 296． 3 -e
Ribavirin 1164． 1 1． 71 680． 8 5． 75 202． 5
aCC50:50% cell toxicity concentration determined by MTT assay．
b Mean of CPE inhibition and NR uptake data．
cEC50:Concentration reducing by 50% the optical density values(or the virus-induced CPE in PC-treated virus-infected cells in relation to virus control)．
The value of EC50 is the mean of three experiments．
dSI:Selectivity Index (CC50 /EC50)．
e-:Lower than 50% inhibitory cytopathic induced by virus．
Conclusions
The results of the present study justify the traditional
therapeutic use of E． densa． These results corroborate
the importance of ethnobotanical surveys for screening
plants as a potential source for bioactive compounds．
The essential oil of E． densa may be applied clinically
for bacteria and fungi infections，e． g．，Staphylococcus
aureus and Candida albicans． Hence these could result
in discovery of novel antimicrobial agents．
Further isolation and identification of the antimicrobial
compounds in the essential oil are needed． Although
from this study it is evident that the essential oil is
showing promising antimicrobial activity，but the meth-
od of action of the essential oil is unknown． Theses ob-
servations may promote a further development of our re-
search in this field．
Based on the test data，further chemical and pharmaco-
logical investigations may be recommended for E． den-
sa．
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Vol. 24 LIU Yi，et al:Chemical Composition，Antimicrobial and Antiviral
Activities of the Essential Oil of Elsholtzia densa Benth．
Acknowledges
The authors are grateful to Dr． Yuele Pan，Institute of
Medicinal Plant Development，Chinese Academic of
Medical Science，Peking Union Medical College，Dr．
Yuhuan Li，Institute of Medicinal Biotechnology，Chi-
nese Academic of Medical Science and Peking Union
Medical College，Dr． Yuan Liu，Analytical and Testing
Center，Beijing Normal University．
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