全 文 ：第 51 卷 第 3 期
2 0 1 5 年 3 月
林 业 科 学
SCIENTIA SILVAE SINICAE
Vol. 51，No. 3
Mar．，2 0 1 5
doi: 10．11707 / j．1001-7488．20150316
Ｒeceived date: 2014 － 05 － 20; Ｒevised date: 2014 － 12 － 01．
Foundation project: The Kevin O’Shea professor foundation．
* Kevin O’Shea is corresponding author．
不同提取方法山苍子油的化学成分与
抗氧化活性分析*
李 芳1 Yaru Song2 Howard Holness2 Kevin O’Shea2
(1． 福建农林大学植物保护学院 福州 350002; 2． 佛罗里达国际大学化学与生物化学系 佛罗里达州 迈阿密 33199)
摘 要: 【目的】比较不同提取方法对山苍子油提取效率与抗氧化活性的影响，通过 GC-MS 分析，研究山苍子油
成分构成与抗氧化活性的内在关联，分析山苍子油抗氧化活性的主要物质基础。【方法】采用液液萃取(分别加以
超声波与磁化辅助处理)、二氧化碳超临界萃取以及水蒸气蒸馏法提取山苍子油，DPPH 法比较不同提取方式对山
苍子油抗氧化活力的影响，GC-MS 分析山苍子油的主要成分，并以 1，4 －二溴苯为内标，采用面积归化法解析主要
成分的相对含量。【结果】山苍子油具有显著的抗氧化活性。液液萃取法的提取效率与山苍子油抗氧化活性最
高，超声波辅助处理油得率最高，为 26. 3%，磁化辅助处理抗氧化活性最优，达到 31. 22 mg·mL － 1 ( IC50 )，二氧化碳
超临界萃取法次之，抗氧化活性为 56. 95 mg·mL － 1 ( IC50 )，水蒸气蒸馏法抗氧化活性最弱，为 64. 95 mg·mL
－ 1
( IC50 ); GC-MS 分析检测出 122 种以上的化合物，包括 12 种脂肪酸，16 种萜烯，18 种含氧萜烯以及其他微量化合
物包括烯烃、醇、酮类与烷烃等。GC-MS 分析结果表明:山苍子干果主要成分为饱和脂肪酸(月桂酸等)、不饱和脂
肪酸(9，12 －十八碳二烯酸、9 －十八碳烯酸等)以及 α 蒎烯、β 蒎烯、柠檬烯等萜烯、氧化萜烯类物质。液液萃取法
得到的山苍子油脂肪酸含量 ( 53. 51% ～ 66. 61% )明显高于二氧化碳超临界萃取 ( 17. 64% ) 与水蒸气蒸馏法
(8. 1% )，而二氧化碳超临界萃取与水蒸气蒸馏法得到的萜烯、氧化萜烯类含量分别达到 29. 37%和 17. 69%，明显
高于液液萃取法(6. 49% ～ 9. 75%)。【结论】磁化辅助处理有利于抗氧化活性物质的提取，且不饱和脂肪酸的含
量最高; 超声波辅助处理可以提升油的得率; 水蒸气蒸馏法和超临界流体萃取法更有利于萜烯类化合物的获取;
联系到水蒸气蒸馏法得到的山苍子油富含萜烯化合物却显示较低的抗氧化活性(64. 95 mg·mL － 1 )，色谱级的柠檬
醛抗氧化活性最低(74. 33 mg·mL － 1 )，因此推测脂肪酸成分(特别是不饱和脂肪酸)是山苍子油抗氧化活性的主要
物质基础。
关键词: 山苍子; GC-MS; 提取; 抗氧化活性
中图分类号: TS227; O657. 63 文献标识码:A 文章编号:1001 － 7488(2015)03 － 0124 － 08
Chemical Component and Activity Analysis of Litsea cubeba Extracts Obtained by
Different Extraction Methods
LI Fang1 Yaru Song2 Howard Holness2 Kevin O’Shea2
(1 ． College of Plant Protection，Fujian Agricultural and Forestry University Fuzhou 350002;
2 ． Department of Chemistry and Biochemistry，Florida International University Miami，FL，USA 33199)
Abstract: 【Objective】The objective of this study is to evaluate the effect of three common methods on the DPPH radical
scavenging activity and the rate of extraction of Litsea cubeba from the dry fruit． GC-MS analysis was carried out to perform
the correlation between chemical composition and antioxidant activity of extracts． 【Method】The antioxidant extracts of
Litsea cubeba were obtained using liquid-liquid extraction (with ultrasonic assisted or magnetic assisted)，SFE (CO2 super-
critical fluid extraction) and steam distillation extraction． The antioxidant activities of the extracts were measured by a free
radical scavenging method using 1，1-diphenyl-2-picrylhydrazyl (DPPH) ． The GC-MS analysis of Litsea cubeba extracts
were carried out with added 1，4-dibromobenzene which served as an internal standard． Peak area normalization was used
to get the relative amount of main compounds in the respective extracts．【Ｒesult】Ｒesults demonstrate that the oil has
significant antioxidant activity． The natural antioxidants are most effectively extracted by employing liquid – liquid
第 3 期 李 芳等: 不同提取方法山苍子油的化学成分与抗氧化活性分析
extraction． The ultrasonic assisted liquid-liquid extraction exhibiting the highest oil yields of 26． 34%，while magnetic
agitation extraction showed highest antioxidant activity of 31． 22 mg·mL － 1 ( IC50 ) ． The antioxidant activity of supercritical
carbon dioxide extraction is 56． 95 mg·mL － 1 ． Steam distillation showed lowest antioxidant activity of 64． 95 mg·mL － 1 ．
More than 122 compounds were identified by GC-MS analysis，including 12 kinds of fatty acids，16 kinds of terpenes，18
kinds of oxygenated terpenes，and other trace compounds including alkenes，alcohols，ketones，and alkanes from these
extracts． GC-MS results show that the main components of the oil of Litsea cubeba from dried fruit are saturated fatty acid
such as lauric acid，unsaturated fatty acids such as 9-Octadecenoic acid，9，12-Octadecadienoic acid and various terpenes
and oxygenated terpenes such as α-pinene，β-pinene， limonene and citral． The fatty acid content of liquid-liquid
extraction was 53． 51% － 66． 61%，apparently higher than 17． 64% of CO2 supercritical fluid extraction (SFE) and 8． 1%
of steam distillation． Meanwhile，the steam distillation and SFE get abundant terpenes and oxygenated terpenes with 29．
37% and 17． 69%，respectively，which were apparently higher than liquid-liquid extraction with 6． 49% － 9． 75% ．
【Conclusion】Liquid-liquid extraction with magnetic assisted extraction made a apparently positive affect on the antioxidant
activity and the highest compounds of unsaturated fatty acid． Meanwhile，ultra-sonic assisted enhance the yield of oil． The
oil extracted by steam distillation rich in terpene compounds and showed lower antioxidant activity (64． 95 mg·mL － 1 ) ．
In addition，HPLC grade citral exhibited the lowest antioxidant activity of IC50 values of 74． 33 mg·mL
－ 1 ． Thus，we can
make a legitimate inference that antioxidant biological activity has been mainly attributed from fatty acid composition ( in
particular unsaturated fatty acid) rather than terpenes or oxygenated terpenes．
Key words: Litsea cubeba; GC-MS; extraction; antioxidant activity
Natural products such as plant extracts，provide
unlimited opportunities for new drug discoveries
because of the unmatched chemical diversity． This has
captured the interest of many researchers to explore
local medicinal plants for valuable medicinal traits．
Antioxidants are substances that protect against
damaging oxidative processes in biological and
environmental systems， inhibiting the harmful chain
reactions initiated by free radicals and other oxidative
processes ( Halliwell et al．，1991 ) ． Naturally active
antioxidants can play major roles in health promoting
activities ( Braga et al．，2003; Barjaktarovic et al．，
2005; Carvalh et al．，2005; Hwang et al．，2005 ) ．
Litsea cubeba contains a range of active substances with
high therapeutic potential(Bighelli et al．，2008; Blois，
1958; Jiang et al．，2009) such as antimicrobial (Wang
et al．，2010)，anticancer(Ho et al．，2010)，antioxidan
and insecticidal activities ( Park et al．，2007; Amer
et al．，2006; Pumnuan et al．， 2010; Feng et al．，
2009) ． Because Litsea cubeba has biological functions
and potential applications，it has become attractive to
researchers． A number of reports have been previously
published on the extraction of the essential oils from
Litsea cubeba(Yang et al．，2010; Zhao et al．，2010;
Wang et al．，1999) ． Previous researchers also reported
the qualitative and quantitative analysis on essential
oils composition from L． cubeba．
Although there are a considerable number of
reports on L． cubeba extraction and biological activity，
only a limited number of studies appear on the most
efficient methods to obtain antioxidant fractions and the
correlation between chemical composition and
antioxidant activity of extracts． Biological antioxidant
capacity of natural product extracts mainly depends on
their composition and their combined action，various
biological activities may be derived from different
extraction methods ( Huang et al．， 2005 ) ． In this
context，the objective of this work is to compare the
chemical compositions derived from different extracted
methods and identify a reliable and practical method for
the liberation of desired natural antioxidants from L．
cubeba．
The antioxidant extracts of L． cubeba were
obtained using liquid-liquid extraction， SFE ( CO2
super-critical fluid extraction ) and steam distillation
extraction． The chemical compositions were determined
by using gas chromatography-mass spectrometry ( GC-
MS ) ． The antioxidant activities were measured by
scavenging activity against 1， 1-diphenyl-2-
picrylhydrazyl (Ho et al．，2010)，The IC50 value was
calculated from the results．
521
林 业 科 学 51 卷
1 Methods and materials
1. 1 Material
Dry fruits of L． cubeba were purchased from
Jianmin drugstore in Fuan city of Fujian Province．
1. 1. 1 Apparatus An ISCO SFXTM super-critical fluid
extractor from Teledyne ISCO (Lincoln，NE，USA) ． UV
and visible spectrum-photometer CAＲY100 bio-VAＲIAN
EL05063009 was used to measure the absorbance for the
DPPH test．
1. 1. 2 Standards，reagents，and solvents Dichloromethane
was of AＲ grade supplied from Fisher Scientific，Atlanta，GA，
USA． Citral (96%) mixture of cis and tran，1，1-diphenyl-2-
picrylhydrazyl ( DPPH ) and 1，4-Dibromobenzene were
HPLC grade purchased from Sigma-Aldrich． ( Sigma
Aldrich，St． Louis，MO，USA) ． Butylated hydroxyanisol
( artificially synthesized antioxidant，analytical standard)
were produced by sigma-aldrich (Sigma，USA) ．
1. 2 Method
1. 2. 1 Preparation of samples 1 ) Liquid-liquid
extraction with ultra-sonic assistance Five grams of dry
L． cubeba seeds were ground to a powder． The powder was
taken up in 20 mLs of CH2Cl2 and 10 mL of saturated
NaCl solution in a 100 mL breaker， employing an
ultrasonic probe to disrupt the cell structures of the plant．
The beaker containing the mixture was subject to
ultrasonic treatment for 10 min． The resulting slurry was
digested for 3 days in room temperature(20 ℃ ) ． Using a
separating funnel， the lower organic solvent layer was
extracted and condensed to get the crude oil．
2) Liquid-liquid extraction Five grams of dry L．
cubeba seeds were ground to a powder，20 mL of CH2Cl2
was added followed by 10 mL of saturated NaCl solution in
a 100 mL beaker． The resulting slurry was digested for 3
days in room temperature ( 20℃ ) ． Using a separating
funnel the lower organic solvent layer was extracted and
condensed to get the crude oil．
3) Liquid-liquid extraction with magnetic assistance
Five grams of dry Litsea cubeba seeds were ground to a
powder，20 mL of CH2Cl2 was added followed by 10 mL of
saturated NaCl solution in a 100 mL beaker． The breaker
was placed above a U-shape magnetization iron and
allowed to digest inside the magnetic field for 3 days．
Using a separating funnel the lower organic solvent layer
was extracted and condensed to get the crude oil．
4) Steam distillation extraction The 100 g powder
of crushed seed of L． cubeba plant materials were placed in
a flask (2 L) together with double distilled water (1. 5
L) ． The mixture was boiled for 4 hours， followed by
exhaustive extraction of the distillate with CH2Cl2，The
extract was condensed in cooling vapor to collect the
essential oil．
5) SFE Five grams of dry L． cubeba seeds were
ground to a powder and placed inside a supercritical fluid
extractor vessel． The powder was extracted at a pressure of
31 MPa and a temperature of 100 ℃ for one hour to get
the essential oil．
The overall yield of oil was calculated as Y (% ):
where Y = mass of extracts / mass of crushed seed × 100．
All essential oils were stored at a temperature of － 4 ℃
until used for the analysis
1. 2. 2 GC-MS analysis The dichloromethane
solutions of the extracts were analyzed by GC-MS． GC-MS
samples was prepared by taking a 100 μL aliquot of the
oil． The oil was dried over anhydrous sodium sulphate
prior to GC-MS analysis． To this was added 5 μL of 1，4-
Dibromobenzene (20 mg·mL － 1 ) solution which served as
an internal standard and 900 μL CH2Cl2 ( reagent grade)
to make up 1mL GC-MS tested sample． GC column was a
DB-5 length 30 m，diameter 0. 25 mm，phase thickness
0. 25 μm． GC Oven parameters were initial temperature of
40 ℃ and held for 1 min followed by a 10 ℃·min － 1 ramp
to 300 ℃ and held for 1 min，inlet was maintained at
280 ℃ ． Mass spectrometer was electron impact at 70 eV
in full scan mode from 50 to 550 amu．
1. 2. 3 Identification method Compounds were
identified by comparison of their mass spectra with those of
the NIST 98 mass spectral database． The components were
analyzed and identified with GC-MS and relative index
(ＲI value) in the same column compared with standard
mixtures of authentic 1， 4-Dibromobenzene． For
quantification，relative peak area percentages were used
and normalized with the internal standard ( accurate 1，4 －
Dibromobenzene added and response peak) to obtain the
relative content of major component without the use of
correction factors． Peak area normalization was used to get
the relative amount of main compounds in the respective
extracts．
1. 2. 4 Antioxidant activity measurement The
extracted oils were diluted in pure methanol ( reagent
grade ) giving a range of 100， 50， 25， 12. 5，
6. 5 mg·mL － 1 ． 0. 1 mL of each dilution was placed in a test
621
第 3 期 李 芳等: 不同提取方法山苍子油的化学成分与抗氧化活性分析
tube in duplicate． The reaction was initiated by addition of
1. 9 mL DPPH solution (51. 54 mg·L －1 in methanol) ． Using
a UV-Vis spectrophotometer，the absorbance was read at 517
nm until the reading reached a plateau． IC50 value was
determined from the plotted graph of scavenging activity
versus the concentration of essential oils，which was defined
as the total antioxidant necessary to decrease the initial
DPPH radical concentration by 50% ．
The DPPH radical scavenging activity was calculated
with the following formula: DPPH radical scavenging
activity (% ) =［A0 － (A1 － A S)］/A0 × 100． Where A0 is
the absorbance of the control solution containing only
DPPH after incubation; A1 is the absorbance in the
presence of plant extract in DPPH solution after
incubation; and A S is the absorbance of sample extract
solution without DPPH for baseline correction arising from
unequal color of the sample solutions ( optical blank for
A1 ) ． The higher the DPPH radical scavenging activity，
the more powerful the antioxidant activity of sample which
results in a lower IC50 value ( Blois， 1958 ) ． For
comparison a sample of Butylated hydroxyanisol (BHA) a
man made antioxidant commonly added to food was also
analyzed by DPPH to reveal its IC50 value using this
method．
2 Ｒesults
Tab. 1 shows the oil yields and DPPH radical
scavenging rates based on the different extraction
methods used． The yellow or coffee colored essential
oil obtained in yields from 0. 30 to 26. 3%， The
ultrasonic assisted liquid-liquid extraction exhibiting
the highest oil yields of 26. 34% with higher
antioxidant activity of 32. 16 mg·mL － 1 IC50 value．
Two-fold dilutions of all crude essential oil
extracts were made with methanol and tested from a
starting dilution 100 to 6. 25 mg·mL － 1 ． There was a
positive correlation between radical scavenging rate and
the concentration of the essential oil． Tab. 1 also shows
the positive influence of ultrasonic or magnetic agitation
extraction on antioxidant activity．
Tab． 1 The DPPH radical scavenging activity on rate of extraction with different method
Extraction method
Yield of crude oil
(W /W) (% )
Ｒegression equation
IC50 /
(mg·mL － 1 )
Extracts colour Ｒ2
Liquid-liquid extraction with ultra-sonic assisted 26. 3 Y = 0. 79x + 24. 51 32. 16 Coffee 0. 914
Liquid-liquid extraction 18. 4 Y = 0. 78x + 21. 33 36. 95 Coffee 0. 917
Liquid-liquid extraction with magnetic assisted 19. 4 Y = 0. 74x + 25. 82 31. 22 Coffee 0. 976
Steam distillation extraction 0. 26 Y = 0. 75x + 1. 35 64. 95 Yellowish 0. 975
SFE 0. 69 Y = 0. 51x + 210 166 56. 95 Light yellow 0. 909
Standard 96% authentic Citral Y = 0. 64x + 2. 25 74. 33 Transparent 0. 948
Butylated hydroxyanisol (BHA) Analytical standard Y = 3. 687 9x + 45. 628 1. 185 4 Transparent 0. 961 4
Tab． 2 showed probable identity of substances and the
relatives percents obtained in GC-MS analysis on extracts
obtained using different extraction methods． The retention
times of Citral (mixture of cis and trans) stayed are 11. 06
( trans) and 11. 47 ( cis) minutes． Internal standard 1，4
dibromobenzene had a retention time of 10. 49 minutes．
Ｒetention times obtained were from 6. 26 － 32. 79 min，
indicating more than 122 identified compounds including
12 kinds of fatty acids，16 kinds of terpenes，18 kinds of
oxygenated terpenes，and other trace compounds including
alkenes，alcohols，ketones，alkanes from these extracts．
The observed variations in the profile and amounts of
individual components have resulted from the variations in
the extraction methods used．
As can be seen in Tab． 2， the composition of the
crude oil obtained using liquid-liquid extractions were
primarily of fatty acids (53. 51% － 66. 61% )，including
saturated fatty acids such as dodecanoic acid (26. 99% －
28. 55% )， monounsaturated fatty acids such as 9-
octadecenoic acid ( 10. 47% － 14. 98% )， and 9，12-
octadecadienoic acid (3. 63% － 7. 31% ) ． Ultrasonic or
magnetic agitation general extracted higher fatty acid yields
up to 62. 84% and 66. 61% respectively． A higher yield
of terpenes ( 8. 47% ) and oxygenated terpenes
(20. 90% ) were obtained from steam distillation，namely
D-limonene ( 5. 52% )， eucalyptol ( 4. 56% )， citral
(4. 05% ) and 1，6-octadien-3-ol (3. 54% ) ． Meanwhile，
SFE extraction yields 17. 30% of saturated fatty acids，
0. 34% of monounsaturated fatty acids， 3. 86% of
terpenes and 13. 83% of oxygenated terpenes．
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林 业 科 学 51 卷
Tab. 2 Probable identity of substances and the relatives percents obtained in GC-MS analysis
Substances
Molecular
Formula
Molecular
Weight
Ｒelative composition on extraction method (% )
(1) (2) (3) (4) (5)
Similarity
(% )≥
1 Dodecanoic acid C12 H24 O2 200. 32 28. 55 26. 99 27. 08 2. 95 11. 55 93
2 n-Decanoic acid C12 H24 O2 200. 32 7. 97 7. 50 8. 61 4. 30 5. 27 94
3 Tetradecanoic acid C14 H28 O2 228. 36 0. 48 0. 64 7. 80 0. 65 0. 24 98
4 N-Hexadecenoic acid C16 H32 O2 256. 43 5. 63 4. 11 0. 29 / / 95
5 Tridecanoic acid C13 H26 O2 214. 34 0. 43 / / / 0. 24 93
6 Octadecadienoic acid C18 H30 O2 278. 43 / / 0. 53 / / 42
Total of saturated fatty acid 43. 06 39. 24 44. 31 7. 90 17. 30
1 9-Octadecenoic acid C18 H34 O2 282. 46 13. 98 10. 47 14. 98 / / 98
2 9，12-Octadecadienoic acid C18 H32 O2 280. 45 5. 68 3. 63 7. 31 / / 97
3 9-Hexadecenoic acid C16 H30 O2 254. 41 0. 12 / / / / 42
4 Trans-2-undecenoic acid C11 H20 O2 184. 28 / 0. 17 0. 01 / / 45
5 2-Myristynoic acid C14 H24 O2 224. 34 / / / 0. 20 / 52
6 10，12-Octadecadiynoic acid C18 H28 O2 276. 41 / / / / 0. 34 48
Total of unsaturated fatty acid 19. 78 14. 27 22. 30 0. 20 0. 34
1 D-limonene C10 H16 136. 23 1. 50 1. 80 1. 52 5. 52 1. 55 95
2 Camphene C10 H16 136. 23 0. 10 0. 14 0. 11 0. 33 0. 06 94
3 β-Pinene C10 H16 136. 24 0. 25 0. 32 0. 33 0. 76 / 97
4 α-Pinene C10 H16 136. 24 0. 33 0. 39 / 0. 25 / 97
5 Bicyclo［3. 1. 0］ hexane C10 H16 136. 24 0. 38 0. 46 0. 46 0. 86 0. 35 91
6 β-Myrcene C10 H16 136. 24 0. 07 0. 15 0. 13 0. 47 59
7 Bicyclo［3. 1. 1］ heptane C10 H16 136. 24 / / / 0. 28 / 49
8 1，4，9-Decatriene C10 H16 136. 23 0. 52 / / / / 47
9 3-Carene C10 H16 136. 23 / / / / 1. 20 43
10 Bicyclo［3. 1. 0］ hex-2-ene C10 H16 136. 23 0. 38 / / / 0. 35 91
12 Copaene C15 H24 204. 35 0. 12 0. 06 0. 14 / / 96
13 Ylangene C15 H24 204. 35 / 0. 15 / / / 45
14 α-Caryophyllene C15 H24 204. 35 0. 03 4. 11 0. 57 0. 35 80
15 1，6，10-Dodecatriene C15 H24 204. 35 / / 0. 13 / / 47
16 Bicyclo［7. 2. 0］ undec-4-ene C15 H24 204. 35 0. 32 0. 09 0. 31 / / 55
Total of terpene 4. 00 7. 67 3. 70 8. 47 3. 86
1 Cis-citral C10 H16 O 152. 24 0. 13 0. 08 0. 09 1. 74 2. 29 99
2 Trans-Citral C10 H16 O 152. 24 0. 24 0. 17 0. 18 2. 31 3. 77 96
3 Caryophyllene oxide C15 H24 O 204. 36 / 0. 56 0. 57 0. 50 2. 32 87
4 Camphor C10 H16 O 152. 24 / / / 0. 49 / 94
5 1，5，7-Octatrien-3-ol C10 H16 O 152. 24 / / / 0. 07 / 45
6 Bicyclo［3. 1. 1］ hept-3-en-2-ol C10 H16 O 152. 25 / / / 0. 44 / 43
7 3-Cyclohexen-1-ol C10 H18 O 154. 25 / / / 1. 43 / 49
8 1，6-octadien-3-ol C10 H18 O 154. 25 0. 30 0. 05 0. 28 3. 54 1. 95 80
9 Cis-beta-terpineol C10 H18 O 154. 25 0. 11 0. 01 0. 12 / / 84
10 Eucalyptol C10 H18 O 154. 25 0. 46 0. 52 0. 41 4. 56 0. 48 98
11 Cis-beta-terpineol C10 H18 O 154. 25 / 0. 01 0. 12 / / 49
12 Terpineol C10 H18 O 154. 25 0. 11 0. 12 0. 08 0. 56 / 68
13 2，6-Octadien-1-ol C10 H18 O 154. 25 1. 07 0. 48 0. 84 / 1. 07 72
14 1，6-Octadien-3-ol C10 H18 O 154. 25 / / 0. 28 3. 54 1. 95 80
15 2-Cyclohexen-1-ol C10 H18 O 154. 25 0. 17 0. 08 0. 03 / / 30
16 5，7-Octadien-3-ol C10 H18 O 154. 25 / / / 0. 59 / 38
17 Bicyclo［3. 1. 0］ hexan-3-ol C10 H18 O 154. 25 / / / 0. 86 / 49
18 Isoborneol C10 H18 O 154. 25 / / / 0. 27 / 59
Total of oxygenated terpenes 2. 49 2. 08 3. 00 20. 90 13. 83
Total of alcohols 1. 37 1. 00 2. 40 2. 93 1. 67
Total of ketone 0. 08 3. 86 0. 55 2. 89 5. 28
Total of others alkene 0 3. 80 0. 87 4. 48 1. 90
Total of alkane 3. 73 1. 86 5. 99 0. 83 6. 78
As shown in Tab． 1，2，3，liquid-liquid extractions
obtained a higher proportion of fatty acids in qualitative
and quantitative composition，while more abundant odor-
activevolatile compounds such as terpenes，alkenes and
oxygenated terpenes were detected from the steam
distillation (method 4) and SFE (method 5) ． Both steam
distillation and SFE extraction were rich in terpene
compounds which showed lower antioxidant activity ( IC50
values of 64. 95 and 56. 95 mg·mL － 1 respectively ) in
comparison to the liquid-liquid extracts． Meanwhile shown
in Tab． 1， HPLC grade citral exhibited the lowest
antioxidant activity of IC50 values of 74. 33 mg·mL
－ 1 ．
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第 3 期 李 芳等: 不同提取方法山苍子油的化学成分与抗氧化活性分析
Thus，taken together，this study concludes that extraction
of substances with the greatest antioxidant properties is
enhanced by ultrasonic and magnetic mechanisms．
Tab． 3 Ｒelatives content of main composition obtained in GC-MS analysis
Ｒt Main compound
Ｒelative content(mg·mL － 1 ) to 1，4-Dibromobenzene
Liquid-liquid extraction 1，2，3
1． Ultrasonic
treat
2． No assisted
treat
3． Magnetic
treat
4． Steam
distillation
5． SFE
6. 26 α-Pinene 0. 003 0. 003 4 / 0. 006 35 /
6. 55 Camphene 0. 000 91 0. 001 3 0. 001 7 0. 008 4 0. 000 21
7. 02 β-Pinene 0. 002 3 0. 002 75 0. 005 0 0. 001 9 /
7. 15 β-Myrcene 0. 000 34 0. 001 3 0. 002 0 0. 001 2
7. 84 D-limonene 0. 013 7 0. 015 5 0. 023 03 0. 014 02 0. 005 4
13. 04 Copaene 0. 001 1 0. 000 52 0. 002 1 / /
13. 04 Ylangene / 0. 001 3 / / /
13. 65 Caryophyllene 0. 000 27 0. 035 3 0. 008 6 / 0. 00123
Total of main terpene 0. 021 62 0. 060 0 0. 042 5 0. 031 87 0. 006 8
7. 91 Eucalyptol 0. 004 2 0. 004 5 0. 006 21 0. 011 6 0. 001 68
8. 51 Terpineol 0. 002 0 0. 001 0 0. 003 03 0. 001 42 /
8. 93 1，6-octadien-3-ol 0. 002 7 0. 000 43 0. 004 242 0. 009 0 0. 006 8
9. 77 Camphor / / / 0. 001 24 /
10. 02 Bicyclo［3. 1. 0］ hexan-3-ol / / / 0. 002 2 /
10. 25 2-Cyclohexen-1-ol 0. 001 5 0. 000 7 0. 000 45 / /
11. 06 Cis-citral 0. 001 2 0. 000 69 0. 001 4 0. 004 42 0. 008 03
11. 47 Trans-Citral 0. 002 2 0. 001 46 0. 002 7 0. 005 9 0. 013 23
12. 86 5，7-Octadien-3-ol / / / 0. 001 5 /
14. 11 1，6-Octadien-3-ol / / 0. 004 242 0. 009 0 0. 006 84
15. 70 Caryophyllene oxide / 0. 004 81 0. 008 6 0. 001 27 0. 011 65
16. 61 1，5，7-Octatrien-3-ol / / / 0. 000 2 /
26. 03 2，6-Octadien-1-ol 0. 009 7 0. 004 1 0. 012 73 / 0. 00375
Total of main oxygenated terpenes 0. 023 5 0. 017 7 0. 045 43 0. 047 75 0. 051 98
12. 75 N-decanoic acid / 0. 064 4 0. 130 4 / 0. 0185
15. 21 n-Dodecanoic acid 0. 072 6 / / 0. 011 /
15. 27 Dodecanoic acid 0. 260 1 0. 230 9 0. 410 3 0. 007 5 0. 040 5
16. 11 z-7-Tetradecanoic acid` 0. 000 5 0. 002 5 / / /
17. 49 Tridecanoic acid 0. 003 9 / / / 0. 000 84
19. 47 Hexadecenoic acid / / 0. 004 4 / /
19. 73 N-Hexadecenoic acid 0. 051 3 0. 035 3 / / /
19. 74 Tetradecanoic acid 0. 003 9 0. 003 0 0. 118 2 0. 001 7 0. 008 4
23. 06 Octadecadienoic acid / / 0. 008 0 / /
Total of main saturated fatty acids 0. 396 8 0. 336 1 0. 671 3 0. 010 3 0. 068 24
7. 21 2-Myristynoic acid / / / 0. 000 51 /
10. 34 10，12-Octadecadiynoic acid / / / / 0. 001 2
15. 09 9-Hexadecenoic acid 0. 001 1 / / / /
22. 44 9，12-Octadecadienoic acid 0. 051 7 0. 031 2 0. 110 7 / /
22. 57 9-Octadecenoic acid 0. 127 4 0. 089 9 0. 226 9 / /
Total of main unsaturated fatty acid 0. 180 2 0. 121 1 0. 337 6 0. 000 51 0. 001 2
3 Conclusion and discussion
The results obtained in the present study showed that
ultrasonic or magnetic assisted liquid-liquid extraction has
advantages that include simplified equipment usage，
higher extraction rates and antioxidant activity，with the
antioxidant activity mainly related to the complex variety of
fatty acids and terpenes within extracts．
Litsea cubeba seeds are known to contain
monoterpenes ( C10 H16 )， sesquiterpenes ( C15 H24 )，
oxygenated terpenes ( such as citral )， alkenes and
unsaturated fatty acids ( such as 9-Octadecenoic acid，9，
12-Octadecadienoic acid )，which are rich in with the
majority of these possessing unsaturated dou

ble bonds of C
C which are the main reasons for their biological activity
of natural extraction．
Steam distillation requires low-cost equipment，and
921
林 业 科 学 51 卷
as a result is more available in rural areas where L． cubeba
is grown and harvested． Steam distillation with higher
temperatures will increase the extraction rate of small
volatile compounds such as mono-terpenes and oxygenated
terpenes． However， long extraction times at elevated
temperatures may destroy some thermo-sensitive
compounds or others compound with high molecule weight
such as fatty acids shown in Tab． 3．
According to the principle of similarly dissolved
solutions，the solubility rate of the non-polarity fraction
will be enhanced in a similar non-polarity solvent． Carbon
dioxide is an ideal solvent for the extraction of natural
products which are normally non-polar． However，its non-
polarity means that polar compounds may only be slightly
soluble in CO2，which is the main reason to explain the
Supercritical carbon dioxide extraction showing weak
antioxidant activity (Tab． 1)，due to poor extraction of the
more polar antioxidant compounds such as unsaturated
fatty acids． Despite a lengthy extraction time，the liquid-
liquid extraction is advantageous to obtain both polar and
non-polarity compounds． Ultrasonic assistance resulted in
additional disruption of the plant cells that lead to higher
overall oil yields and higher antioxidant activity of
essential oil．
The results obtained in the present study revealed
that liquid-liquid extraction with magnetic assisted
extraction (method 3) made a significantly positive affect
on the antioxidant activity and yield of the main active
compounds， particularly unsaturated fatty acid． In
addition，unsaturated fatty acids，such as 9-octadecenoic
acid，are well-known as natural active components that
show significant antioxidant activity． Thus， making a
legitimate inference that antioxidant biological activity has
been mainly attributed from fatty acid composition rather
than terpenes or oxygenated terpenes．
The enhancement observed with magnetic assistance
may be attributed to the magnetic field as it has been
reported that an energy field can change the microstructure
of biomembrane， thus enhancing the substances diffuse
rate ( Gribova et al．，2008; Cosio et al．，2006 ) ． This
magnetic effect was in accordance with that previously
published and states that natural antioxidants are most
completely extracted by maceration in a constant electric
field ( Gribova et al．，2008 ) This finding indicates that
magnetic-effect extraction is an acceptable technique for
in-depth studies for improving the extraction of substances
with antioxidant capacity from L． cubeba．
Some previous studies have reported that Litsea
cubeba have significant antioxidant properties and that the
essential oil from the fresh fruit contains 75% citral
(Deng et al．，2011) ． The present study did not produce
these results． However，this could be explained in terms
of attributed to a significance factors the citral mainly
come from fresh fruit peel，while fatty acid composition
primarily from dry fruit kernel (Ho et al．，2010; Duan et
al．，2010) ．
Acknowledgements
The work was supported by the Florida International
University，Department of Chemistry and Biochemistry，
special thanks to Dr． Kenneth G． Furton for use of his
supercritical fluid extractor．
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