全 文 ：第29卷第5期　　　　　　　　 　西南 大学学 报 (自然科学版)　　　　　　　　　　　2007年 5月
Vo. 29　No. 5 Journal of Southw est Unive rsity (N atural Science Edi tion) May　2007
文章编号:1000 2642(2007)05 0122 06
A Study of the Composition of Essential
Oils from Emblica (Phyllanthus emblica L.)
Fruit by Supercritical Fluid Extraction
and Their Antioxidant Activity
①
LIU Xiao-li , 　ZHAO Mou-ming , 　CUI 　Chun , 　LUO 　Wei
College of Light Industry and Food Science , South China University of Technology , Guangzhou Guangdong 510640 , China
Abstract:Experiments we re conducted to investiga te the components and in vitro antio xidant activity o f the essential
oils from emblica(Phy l lanthus emblica L.) fruit. The essential o ils were obtained by supercritical fluid ex trac tion
(SFE) and an o rthogonal ar ray design(L9) with thr ee facto rs and thr ee levels wa s employed to optimize. Operating
conditions. Optimum ex traction conditions w ere:pressur e 20 MPa , temperature 35℃, ext ract time 3h and me thanol
10 mL. The essential oils w ere analy zed by a ga s chroma to g raph-mass spectro scope and a to tal of 30 compounds ac-
counting fo r 99. 86% of emblica essential oils w ere identified. The majo r components we re β-bourbonene , te traco-
sanol , eugeno l , β-cary ophylleneand , and 2 , 3-dihydroxypropanol , accounting fo r 38. 23%, 17. 20%, 10. 94%,
5. 39% and 4. 36% o f the total , respectively . The antiox idant activities of the oils were evalua ted using 2 , 2-diphen-
y l-1-picry lhydrazyl (DPPH) and lino leic acid per oxidation assay s. The EC50 va lue o f the emblica e ssential oils fo r
scavenging DPPH radica ls(11. 2±1. 6 μg /m L) w as highe r than tha t of BH A andα-to copher ol , w hile the oils exhib-
ited highe r inhibition activity than α-to copher ol in the lino leic acid sy stem. Methyl eugeno l , β-bourbonene and β-
ca ryophy llene might be responsible fo r antio xidant activities. The results suggested that emblica f ruit could be a po-
tential source of antio xidants of plant o rig in.
Key words:Supercritical carbon dio xide ex traction;Essential oil;Antio xidant activity;Phy llanthus emblica L.
CLC number:TS202. 3;TQ028. 3 Document code:A
1　Introduction
Free radicals are an enti re class o f highly reactive molecules de rived from the no rmal metabo lism of ox-
ygen o r f rom enviormental factors and agents. Oxidat ive damage to crucial cellular mo lecules induced by
free radicals has been implicated as a po ssible facto r in the etiolo gy o f seve ral human diseases , including
cancer and cardio vascular disease[ 1] . In recent y ears , there is an increasing interest in f inding antio xidants
f rom phytochemicals , because they can inhibi t the propagation of free radical reactions to pro tect the hu-
man body from diseases and retard lipid oxidat ive rancidity in foods[ 2] . M any researchers have repo rted
① 收稿日期:2006 12 05
基金项目:国家十一五科技支撑计划项目(2006BAD27B03).
作者简介:刘晓丽(1977 ), 女 , 山西大同人 , 博士研究生 , 主要从事食品生物技术的研究.
通讯作者:赵谋明 , 男 , 博士 , 教授 , 博士研究生导师.
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that essential oils exhibi ted ant imicrobial , ant ioxidant and radical-scavenging propert ies. In some cases , a
direct food-related application o f essential oils has been tested[ 3] .
Supe rcritical f luid ex t raction (SFE) can be considered as one of the most potential ly useful methods
fo r sample preparat ion and has an advantage over the conventional methods , e. g. hydrodist illation , steam
distillation and solvent ext raction , in isolating vo latile compounds f rom natural matrices[ 4] .
Emblica (Phy l lanthus emblica L. ) as an euphorbiaceous plant is w idely distributed in subtropical and
tropical areas o f China , India , Indonesia and M alay Peninsula. Emblica f rui t has been used w idely as a lo-
cal t radi tional medicine , such as Chinese herbal medicine and Ayurvedic medicine[ 5] . Emblica f ruit i s repo r-
ted to have antiox idant , hypolipidemic , hypog lycem ic , and hepatoprotective activities[ 6] . To our best
know ledge , the re is no repo rt about the essential oil s in relation to their antio xidant activity of emblica
f ruit . The aims o f this w o rk w ere to evaluate the in v itro ant ioxidant prope rties o f the essential oils of em-
blica f ruit , obtained by supercri tical CO 2 ex traction and then determine the chemical composit ions o f these
oils by gas chromatog raphy-mass spectro scopy (GC-MS).
2　Materials and methods
2. 1　Plant materials and chemicals
Fresh f ruits of emblica (Phy l lanthusemblica L. )we re collected f rom Huizhou , Guangdong pro vince.
DPPH(1 , 1-dipheny l-2-picry lhydrazy l) and lino leic acid (99%)were purchased from Sigma Chemical Co.
(St. Louis , MO , USA). Al l o ther chemicals and reagents used w ere o f analy tical grade.
2. 2　Extraction of the essential oils by supercritical carbon dioxide
Fresh emblica f lesh w as air-dried in an oven at 40 ℃ and then g round to 0. 2(0. 4 mm part icles in a
cut ting mill. Pow der (500 g) was ex t racted by the SFE with CO 2 plus methanol co-solvent (SFE-MeOH)
was conducted in one lit re stainless steel vessels w ith a 1L-SFE (Light Indust ry Research Inst itute , Guan-
g zhou , China) supe rcritical f luid ex t ractor (show ed in Fig. 1). To improve the yield of ex t ractive s , nine
runs of o rthogonal experiment w ere carried out at different pressures (15 , 20 and 25 MPa), temperatures
(25 , 35 and 45 ℃) and t imes (1 , 2 and 3 h). Three dif ferent concentrations of methano l (10 , 30 and
50 mL)were used as modi fier. The CO 2 f low rate w as 2. 5 mL /min in all experiments. The obtained es-
sent ial oil s w ere dried ove r anhydrous sodium sulphate , then fil t rated through 0. 45μm filter paper , and fi-
nally sto red at 4 ℃ fo r further test and analy ses.
(1) CO 2 tube;(2) shu t-of f valve;(3) gas fil ter;(4) coolin g bath;(5) pum p;(6) pressu re regulat ing valve;
(7) pres sure gauge;(8) shut-off valve;(9) ext ractor;(10) w ater b ath;(11) res trictor;(12) collect ion vials w ith ethanol;
(13) f lowmeter;(14) gas collector ;(15) m odif ier pump and (16) ext ract removal valves.
Fig. 1　Schematic Drawing of the Supercritical Extraction Equipment
2. 3　GC–MS analytic conditions for the essential oils
The essent ial oils we re analy zed using a Shimadzu GC-MS sy stem equipped w ith a Q P5050 M ass Spec-
t rometer (Kyo to , Japan) and a DB-5 capillary co lumn (60 m ×0. 25 mm , 0. 25 μm thick , Folsom , CA ,
123第 5期　　　LIU Xiao-l i ,etal :A Study of the Composi tion of E ssential Oi ls f rom Emblica (Phy l lanthus embl ica L.)Frui t by Supe rc ri tical Fluid Ex trac tion and Their Antio xidant Act ivi ty
USA). The oven temperature w as prog rammed from 40 to 300 ℃at a rate o f 10 ℃/min and held fo r 10min
at 300 ℃. The injector temperature and ion source temperature w ere 250 ℃. The M S spect ra w ere scanned
in the mass range of m /z 50 ～ 500 , with the detecto r voltage w as 70 eV. Helium was used as the carrier
gas w hile the column f low rate w as 1. 1 mL /min. The sample (1μL)was injected into the capillary column
with a spli t ratio o f 2∶1. Identif ication of oil compounds w as based on the comparison of their mass spec-
t ra and retention times (RT)w ith those recorded in the NIS T98 mass spect ral library (N ational Institute
of Standards and Technolog ies , USA) and the li terature[ 7] . Quanti tative analy sis of each essential oi l com-
ponent (expre ssed as a percentag es) was carried out by peak area normalization measurement.
2. 4　Determination of antioxidant activity by the DPPH-radical-scavenging method
The free radical scavenging activi ty w as de termined by the method o f S himada , Fujikaw a , Yahara ,
and et al. [ 8] .
2. 5　Determination of antioxidant activity by peroxidation inhibition of the linoleic acid method
The ant ioxidant activity o f emblica essential oils on inhibi tion of linoleic acid pero xidation was assayed
using the thiocyanate me thod[ 9] .
3　Results and discussion
3. 1　The orthogonal experiment determined the best extract condition
The resul ts of pre-experiments show ed that pressure , temperature , ex t ract time and methanol volume
had signif icant ef fect on the yield of the SFE extract. The refore , a three-factor s , three-level (L9) orthogo-
nal ar ray de sign w as employed to optimize the ope rating conditions of the SFE process. The resul t w as
show n in T able 1. T he ext raction y ield ranged betw een 1. 1 and 2. 5%(w /w) depending on ex t raction con-
ditions. The significance of these facto rs affecting the yield w as in the fol lowing o rder:pressures > t ime
> temperature > methanol volume. Optimum ex traction condi tions:pressure 20 MPa , temperature
35 ℃, ext ract time 3h and methano l 10 mL.
Table 1　The Theorthogonal Array Design with four Factors and Three Levels(L9) for Oil Extraction by SCF
Run
Pressure
/MPa
Tem pera tur e
/℃
Time
/h
Me thanol volume
/mL
Yield
(%, w /w)
1 15 25 1 10 1. 1
2 15 35 2 30 1. 2
3 15 45 3 50 1. 5
4 20 25 2 50 1. 8
5 20 35 3 10 2. 5
6 20 45 1 30 1. 9
7 25 25 3 30 2. 0
8 25 35 2 50 2. 2
9 25 45 1 10 1. 6
K1 3. 8 4. 9 4. 0 5. 2
K2 6. 2 5. 9 5. 2 5. 1
K3 5. 8 5. 0 6. 0 5. 5
K1(equa l) 1. 3 1. 6 1. 3 1. 7
K2(equa l) 2. 1 2. 0 1. 7 1. 7
K3(equa l) 1. 9 1. 7 2. 0 1. 8
R 0. 8 0. 4 0. 7 0. 1
3. 2　Chemical composition of the essential oils
The GC-MS profile o f the essential oil s f rom emblica f ruit w as show n in Fig. 2. Thir ty compounds i-
124 西南大学学报(自然科学版)　　　　　　　　　　　　第 29卷
dent ified by GC-MS accounted for 91. 76 % of the essential oils(Table 2). As show n in Table 2 , the majo r
compounds of emblica f ruit oils were β-bourbonene (30. 15%), tet racosanol (14. 42%), tet racosane
(9. 67%), eugeno l(8. 25%), β-cary ophy llene (8. 56%), and heptadecanol (2. 92%).
Table 2　Chemical Composition of the Essential Oils from Emblica Fruit
NO. Compound RT a % RAb
1 Heptane 3. 22 trc
2 1-Octen-3-o l 3. 46 0. 21
3 2 , 3-Dihydroxypropanol 3. 57 2. 25
4 Limonene 5. 49 tr
5 2-Furanone 6. 30 0. 23
6 Undecane 6. 48 0. 19
7 Camphor 6. 97 0. 22
8 β-Neoclovene 7. 38 0. 11
9 Dibuty l phthalate 20. 34 2. 01
10 Decanal 20. 69 1. 56
11 Nerol 26. 30 0. 12
12 Quinoline 26. 52 0. 85
13 2-Undecanone 31. 57 0. 14
14 Thymol 36. 41 0. 78
15 Me thy l eugenol 37. 27 1. 90
16 Eugenol 37. 98 8. 25
NO. Compound RT a % RA b
17 Pentadecanone 40. 00 0. 12
18 3-Hexenol 40. 15 0. 58
19 Eicosanol 42. 97 0. 66
20 Palmitic acid 43. 12 1. 97
21 β-Caryophy llene 46. 02 8. 56
22 Bo rneol 46. 15 0. 45
23 β-Bourbonene 49. 31 30. 15
24 β-Elemene 52. 26 1. 21
25 Heptadecanol 55. 32 2. 92
26 Nonadecane 55. 87 0. 30
27
6 , 10 , 14-T rimethy l-pentadecan-
one
56. 60 0. 75
28 Carpeste rol 58. 18 0. 88
29 Tetr aco sane 60. 20 9. 67
30 Tetr aco sano l 61. 54 14. 42
To tal 91. 76
　　a. RT indica ted the retention time on the DB-5 column in minutes. b. RA indica ted the re lativ e a rea(peak area relative to
the to tal peak area). c. T race(t r)≤0. 09 %.
Fig. 2　A Gas Chromatographic Profile of the Essential Oils of Emblica Fruit
3. 3　Antio xidant act ivity
The antio xidant act ivity o f the essential oils w as evaluated using scavenging DPPH free radicals and
inhibit ion capabi li ty of linoleic acid pe roxidation. Free radical scaveng ing capacity of the essential oils
measured by the DPPH assay w as show n in Table 3. The EC50 values o f the emblica essential oi ls , BHA
andα-to cophe ro l w ere 11. 2±1. 6 , 13. 3±0. 2 and 20. 7±0. 9 μg /mL , respectively. And the antio xidant
activi ty of the emblica essent ial oil s w as signif icant ly different (p <0. 05) f rom that of BHA and α-to-
cophero l.
Table 3　Free Radical Scavenging Capacities of the Essential Oils from Emblica Fruit
Sample s EC50 value (μg /mL)a
Emblica essential oil 11. 2±1. 6A b
BHA 13. 3±0. 2B
α-Tocophe rol 20. 7±0. 9C
125第 5期　　　LIU Xiao-l i ,etal :A Study of the Composi tion of E ssential Oi ls f rom Emblica (Phy l lanthus embl ica L.)Frui t by Supe rc ri tical Fluid Ex trac tion and Their Antio xidant Act ivi ty
　　a. EC50 value w as de te rmined to be the effective concentra tions at which DPPH radicals we re scavenged by 50 %. The
EC50 value w as obtained using a linea r reg ression analy sis.
b. Each value w as expressed as the mean±SD(n=3). Means with different letter s within a column were significantly dif-
fer ent a t p<0. 05.
Fig. 3 display ed the inhibi tion activi ty of emblica essent ial oils and reference antioxidants against lino-
leic acid pero xidat ion. The essential oils show ed a significantly higher (p<0. 05) inhibition activity thanα-
to cophe rol but a low er activi ty than BHA.
—◆—:Blank;—□—:Emblica es sen tial oils;
—△—:BHA;—×—:α-tocoph erol.
Fig. 3　Capability of Linoleic Acid Peroxidation
of the Emblica Essential Oils
Various approaches for the de termination of the
ant ioxidant activi ties of the plant ex t racts have been
int roduced but led to different resul ts , which are rel-
a tively hardly comparable , due to dif ferent mecha-
nisms of ant ioxidant ability used , depending on the
oxidant /antio xidant models employed and lipophilic /
hydrophilic balance[ 10] . Therefo re , the antioxidant
activi ty o f the emblica essent ial oils here w as deter-
mined by tw o complementary test sy stems (DPPH
free radical scavenging and linoleic acid sy stems). A s
show n in Table 3 and Fig. 3 , the emblica essential
oils exhibited good ant ioxidant act ivities in the tw o
test sy stems. Similar results we re obtained wi th the essential oils f rom eucalyptus , black sesame seed and
lippia[ 7] . M ethyl eugenol , β-bourbonene , and β-cary ophy llene w ere considered as one of the principal an-
tio xidative components in these oils. As high contents o f the components in emblica essential oil s exist , it
i s expected that these compounds should play an important ro le in the antioxidant activity of emblica essen-
tial oi ls.
4　Conclusion
A three factors , three level(L 9) o rthogonal array design w as employed to opt imize the operating con-
ditions of the SFE process. The significance o f these facto rs af fecting the yield w as in the fo llowing orde r:
pressures>t ime > temperature > methanol volume. Optimum extract ion conditions:pressures 20MPa ,
temperature 35 ℃, ex t ract time 3 h and methano l 10 mL. The result of gas chromatog raphy-mass spec-
t ro scopy analysis show ed that emblica essential oils obtained by SFE w ere rich in β-bourbonene (30. 15%),
tet raco sano l (14. 42%), tet racosane (9. 67%), eugeno l (8. 25%), β-cary ophy llene (8. 56%), and hepta-
decano l(2. 92%). The EC50 value of emblica essential oil for scavenging DPPH radicals w as 11. 2±1. 6
μg /mL , higher than that of BHA andα-tocopherol. Furthermo re , the oi l exhibited higher inhibi tion act ivi-
ty than α-tocopherol in linoleic acid sy stem. The st rong antiox idant activi ty of the emblica essential oils
sugge sted that the f ruit may be used as a promising source of natural antio xidants. Methy l eugeno l , β-
bourbonene , and β-caryophyllene might be responsible fo r ant ioxidant act ivities. Howeve r , further investi-
gations are needed to determine the relationship be tw een the chemical composition and the antio xidant ac-
tivity of the oils.
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超临界 CO 2 萃取余甘子精油成分及其抗氧化活性研究
刘晓丽 , 　赵谋明 , 　崔　春 , 　罗　维
华南理工大学 轻工与食品学院 , 广东 广州 510640
摘要:采用超临界 CO2 萃取余甘子精油 , 选择萃取压力 、 温度 、时间和甲醇添加量为因素进行正交实验 , 得出影响精油得率
的大小次序为:萃取压力>萃取时间>萃取温度>甲醇添加量 , 优选出超临界 CO2 萃取的最佳工艺条件为:萃取压力
20MPa , 萃取温度 35 ℃, 萃取时间 3 h , 甲醇添加量 10 mL . 在这个最佳条件下试验 , 余甘子精油得率为 2. 5%(w /w). 采用
DPPH 自由基体系和亚油酸过氧化体系测定余甘子精油的抗氧化活性 , 并运用 GC-MS 法分析鉴定其化学成分. 结果表明 ,
超临界CO 2 萃取的余甘子精油具有较强的抗氧化活性 , 其清除 DPPH 自由基的活性优于维生素 E 和合成抗氧化剂 BHA , 抑
制亚油酸过氧化的能力优于维生素 E;从该精油中鉴定出了 30 种化学成分 , 其中主要成分为 β 波旁烯(30. 15%)、 丁香酚
(8. 25%)、 β 丁香烯(8. 56%)、 二十四醇(14. 42%)及十七烷醇(2. 92%), 初步推断甲基丁香酚 、 β 丁香烯 、β 波旁烯为主
要的抗氧化成分. 余甘子精油具有较强的抗氧化作用 , 可望开发成为天然食品抗氧化剂.
关键词:超临界 CO 2 萃取;精油;抗氧化;余甘子
责任编辑　陈绍兰　　　　
127第 5期　　　LIU Xiao-l i ,etal :A Study of the Composi tion of E ssential Oi ls f rom Emblica (Phy l lanthus embl ica L.)Frui t by Supe rc ri tical Fluid Ex trac tion and Their Antio xidant Act ivi ty