全 文 ：第 34 卷第 1 期
2014 年 2 月
林 产 化 学 与 工 业
Chemistry and Industry of Forest Products
Vol． 34 No． 1
Feb． 2014
doi:10． 3969 / j． issn． 0253-2417． 2014． 01． 021
Chemical Composition of Leaf Essential Oil of
Synsepalum dulcificum and Evaluation of
Its Antibacterial and Antitumoral
Activities in vitro
收稿日期:2012-12-27
基金项目:科技部 973 前期研究专项课题(2011CB512010) ;海南省高等学校科学研究基金项目(Hjkj2012-22) ;海南省高等学校优秀
中青年骨干教师基金项目(无编号)
作者简介:卢圣楼(1987—) ，男，江苏南京人，硕士生，从事天然产物化学的研究;E-mail:lushenglou@ 163． com
* 通讯作者:刘 红(1967—) ，教授，博士，硕士生导师，主要研究领域为天然产物分离及保健食品的开发。
LU Sheng-lou
LU Sheng-lou，LIU Hong* ，CHEN Guang-ying，
HAN Chang-ri，ZANG Wen-xia
( College of Chemistry and Chemical Engineering，Hainan Normal University，Key Laboratory of
Tropical Medicinal Plant Chemistry of Ministry of Education，Haikou 571127，China)
Abstract:The aim of this study was to determine the chemical composition of Synsepalum dulcificum leaf
essential oil obtained by steam distillation． The in vitro antibacterial activity and antitumoral activity of
this essential oil was characterized． Sixty-eight compositions were separated． Among them 44 components were identified and
represented 92． 14% of the total detected constituents． The major chemical compounds of leaf essential oil were spathulenol
(24. 194%) ，limonene (15． 805%) ，diisooctyl phthalate (12． 402%) ，dibutyl phthalate (10． 326%) ，palmitic acid
(4. 865%)and linalool (2． 139%)． The result of the antimicrobial assay showed that the essential oil displayed varying degrees
of antibacterial activity against all tested bacterial except for Pseudomonas aeruginosa， along with minimum inhibition
concentration(MIC)values from 39． 06 to 252． 15 mg /L． In addition，the antitumoral activity using MTT assay of the leaf
essential oil exhibited this oil was effective against human K562 cancer cell line in a dose dependent manner． Its inhibition
concentration 50% (IC50)value was 13． 5 mg /L．
Key words:Synsepalum dulcificum leaf;essential oil;chemical composition;antibacterial activity;antitumoral activity
CLC number:TQ351 Document code:A Article ID:0253-2417(2014)01-0121-07
神秘果叶挥发油化学成分分析及抗菌、抗肿瘤活性
卢圣楼，刘 红，陈光英，韩长日，臧文霞
( 海南师范大学化学与化工学院，热带药用植物化学教育部重点实验室，海南 海口 571127)
摘 要:采用水蒸气蒸馏法提取海南产神秘果叶挥发油，利用气相色谱-质谱联用技术( GC-MS) 分析挥发油的化学成分，
并对挥发油的抑菌和抗肿瘤活性进行了评价。结果从挥发油中共分离出 68 个化合物，鉴定出 44 种化学成分，占挥发油
总质量分数的 92． 14%。其中质量分数较高的组分为匙叶桉油烯醇( 24． 194% ) ，柠檬烯( 15． 805% ) ，邻苯二甲酸二异辛
酯( 12． 402% ) ，邻苯二甲酸二丁酯( 10． 326% ) ，棕榈酸( 4． 865% ) 和芳樟醇( 2． 139% ) 。体外抗菌试验的结果表明，神秘
果叶挥发油除了对绿脓杆菌没有明显的抑制作用，对其他 7 个试验菌株均具有较好的抑菌活性，最小抑菌浓度( MIC) 范
围为 39． 06～252． 15 mg /L。抗肿瘤活性试验的结果表明，神秘果叶挥发油能够有效抑制人白血病细胞 K562 的增殖，半
数抑制浓度( IC50 ) 值为 13． 5 mg /L。
关键词:神秘果叶;挥发油;化学成分;抗菌活性;抗肿瘤活性
122 林 产 化 学 与 工 业 第 34 卷
Plant essential oils are valuable natural products used as raw materials in many fields， including
perfumes，cosmetics，aromatherapy，phototherapy，spices and nutrition． Furthermore，the essential oils are
used in traditional medicine for their various activities，such as antimicrobial，antioxidant，antifungal，
antitumoral，spasmolytic，carminative，hepatoprotective and antiviral activities［1］． Synsepalum dulcificum
(Sapotaceae) ，an evergreen shrub indigenous to West Africa，produces red berries containing a glycoprotein，
miraculin，which has the unusual ability to modify a sour taste into a amazingly excellent sweet taste［2－3］．
Subsequently，S． dulcificum was extensively used as an adjuvant for diabetic patients with insulin resistance or
for individuals who need sweeteners instead of sugar［4］． In the course of screening for pharmacological activity
from tropical medicinal plants，S． dulcificum plant was chosen for further phytochemical investigation． The
published results elaborated that S． dulcificum leaf had obvious curative effect on enhancing liver function，
heartburn，indigestion，poor appetite and other symptoms，such as atherosclerosis，high blood pressure and
diabetes［5］． Biochemical constitutes obtained from S． dulcificum stem had antioxidant properties and inhibitory
effects on human melanoma proliferation，and reduced mushroom tyrosinase activity［6］． The protein extracted
from S． dulcificum seed could lower blood glucose and promote the secretion of insulin of diabetic mice［7］．
Ｒecently，S． dulcificum has received special attention as source of natural medicine and dietary food in some
countries［8－9］． The quest for new antimicrobial and chemopreventive drugs based on traditional herbs is of great
interest． However，so far the presence of chemical compounds and bioactivities in S． dulcificum essential oil
has not been demonstrated． Therefore，the aim of the present study was designed to determine the chemical
compounds of leaf essential oil of S． dulcificum as well as to evaluate the antibacterial activity of this essential
oil towards eight microbial strains． Furthermore，we investigate its cytotoxicity activity on a series of human
cancer cell lines， SPC-A-1 (Lung) ，BEL-7402 (Hepatocellular) ，SGC-7901 (Gastric) ，K562
(Myelogenous Leukaemia) ，by means of the MTT (3-(4，5)-dimethylthiazol-2-yl)-2，5-diphenyltetrazolium
bromide)assay． We attempt to find its use of this essential oil for pharmaceutical treatment and natural
therapies．
1 Materials and Methods
1． 1 Plant material
The leaf of S． dulcificum was collected during October 2011 in Baoting county，Hainan Province，China．
A voucher specimen was deposited in the Key Laboratory of Tropical Medicinal Plant Chemistry of Ministry of
Education，Hainan Normal University，China．
1． 2 Microbial strains
The in vitro antibacterial activity of the essential oil was evaluated against panel，which included
laboratory control strains:six Gram-positive bacteria (Bacillus subtilis，Bacillus cereus，Staphylococcus albus，
Staphylococcus aureus，Micrococcus tetragenus ＆ Micrococcus luteus) and two Gram-negative bacteria
(Escherichia coli ＆ Pseudomonas aeruginosa)． The microbial strains were obtained from School of Life
Science，Hainan Normal University． Cultures of each bacterial strain were maintained on Luria-Broth (LB)
medium at 37℃ ．
1． 3 Cells
Cell lines (SPC-A-1，BEL-7402，SGC-7901 ＆ K562)were originally obtained from Shanghai Institutes
for Biological Sciences，Chinese Academy of Sciences and maintained in Ｒoswell Park Memorial Institute-1640
(ＲPMI-1640，Gibco BＲL，USA)medium，supplemented with 10% neonatal bovine serum (NBS) ，0． 2%
NaHCO3，100 IU /mL penicillin，100 mg /L streptomycin and 2 mmol /L L-glutamine at a humidified
第 1 期 卢圣楼，等:神秘果叶挥发油化学成分分析及抗菌、抗肿瘤活性 123
atmosphere containing 5% CO2 at 37 ℃ ．
1． 4 Essential oil extraction
Fresh leaf was chopped and submitted to hydrodistillation for 6 h using a Clevenger-type apparatus． The
oil was recovered with diethyl ether and dried over anhydrous sodium sulfate for 24 h． The solvent was
removed using the rotary evaporator under vacuum． Obtained essential oil was preserved in the sealed brown
glass vials at 4℃ under refrigeration till further analysis．
1． 5 GC-MS analysis
The analysis of volatile compounds was performed using the GC-MS 6890-5975 system (Agilent
Technologies，Palo Alto，CA，USA)equipped with a HP-5MS fused silica capillary column (30 m ×0． 25 mm
i． d．，with 0． 25 μm membrane thickness，Agilent Technologies)． The equipments were conditioned and
programmed according to the required parameters． For GC-MS detection，the electron ionization (EI)system
with ionization energy of 70 eV，the carrier gas was helium at a constant flow rate of 1 mL /min． The front inlet
in split-less mode and the mass transfer line temperature were set at 250 ℃ and 280 ℃，respectively． Diluted
essential oil (1 ∶ 50 in ether，v /v)of 1 μL was injected manually and analyzed with the column held initially
at 80 ℃ for 1 min and then programmed to 150℃ with a 5℃ /min heating ramp，subsequently held at 280 ℃
for 40 min． The major constituents of the essential oil were identified by comparing their mass spectra with the
NIST08 mass spectral database library，and confirmed by comparison with data published in the literature
whenever possible． Ｒelative percentage of each compound in essential oil was expressed as percentage by
integrating the peak area of the spectrograms．
1． 6 Antibacterial activity assay
The paper disc diffusion method was employed to determine the growth inhibition caused by leaf essential
oil from S． dulcificum against the above strains［10］． Briefly，the tested bacterial suspensions were adjusted to
107 CFU /mL and spread in LB medium (10 － 15 mL per  90 mm Petri dishes)using a sterile cotton swab．
Subsequently，filter paper discs ( 6 mm;Whatman #1)were placed on the surface of Petri dishes and
impregnated with 20 μL of the essential oil dissolved in dimethylsulfoxide (DMSO)at 10 g /L． The Petri
dishes were inverted and incubated at 37℃ for 24 h． The zone of growth inhibition diameter against the tested
bacterial was measured to evaluate antimicrobial activity of essential oil． Negative control was set up with
equivalent quantities of DMSO． Antibiotic ampicillin (10． 0 mg /L)was used as positive test controls． All
determinations were performed in triplicate．
The minimum inhibitory concentration (MIC)of the essential oil against the test strains was evaluated by
micro-dilution method with slight modification［11］． The MIC was determined only with micro-organisms that
displayed inhibitory zones． Dilutions were prepared in a 96-well microtitre plates to get final concentration
ranging from 0． 01 to 0． 30 g /L of essential oil in the medium． Finally，100 μL of inoculums were inoculated
on to the microplates． Plates were incubated at 37 ℃ for 24 h． The lowest concentration of tested essential oil，
which did not show any visual growth after macroscopic evaluation，was determined as MIC． A negative control
was also included in the test using a filter paper disc saturated with DMSO to check possible activity of this
solvent against the bacteria assayed． All determinations were performed in triplicate．
1． 7 Antitumoral activity assay
The cytotoxic effects of the essential oil on four human tumor cell lines were assayed by the MTT
assay［12－13］． Essential oil was solubilized in DMSO then diluted with phosphate buffered saline (PBS)for
further use． The cells seeded at a density of 104 ind per well were cultivated for 24 h before the test essential
oil was added． The essential oil (20 μL per well)of different dilutions (100，10 and 1 mg /L)was applied to
124 林 产 化 学 与 工 业 第 34 卷
the wells of a 96-well plate containing confluent cell monolayers (six wells per concentration)． The cells were
cultivated for a further 72 h． The dilution medium without the sample was served as control． The IC50 values
were calculated by linear regression of plots where the abscissa and the ordinate represented the concentration
of tested oil，and the average percent of antitumoral activity from three separate tests，respectively．
1． 8 Statistical analysis
The tests were conducted in triplicate and data from experiments were analyzed by SPSS 14． 0 and
calculated as mean ± SD．
2 Ｒesults and Discussion
2． 1 Chemical composition of the essential oil
The essential oil obtained by steam distillation of the fresh leaf of S． dulcificum afforded an average yield
of 0． 1% on wet weight basis． Freshly isolated essential oil was a straw yellow liquid with intensive and
narcotic odour． The chemical components of the essential oil of S． dulcificum were analyzed by GC /MS． Sixty-
eight compositions were separated． Among them，44 components were identified and represented 92． 14% of
the total detected constituents． Their component name，retention time，molecular formula and percentages
were summarized in Table 1． The complex mixture components of essential oil were separated into six classes．
They were oxygenated sesquiterpenes，monoterpene hydrocarbons，aliphatic esters，oxygenated monoterpenes，
hydrocarbons and others． Spathulenol (24． 194 %)，limonene (15． 805 %)，diisooctyl phthalate (12． 402 %)，
dibutyl phthalate (10． 326 %) ，palmitic acid (4． 865 %) and linalool (2． 139 %)were the major
constituents． They comprised 69． 731% of the essential oil．
The high percentage of spathulenol has been reported to have antiasthmatic and expectorant actions［14］．
Limonene has been found to exhibit against mammary tumors in rats and metastasis of human gastric cancer［15］
and antifungal properties［16］． Linalool is one of the most useful monoterpene alcohols for the perfumery
industry as well as for synthesis route to vitamin E［17］， Furthermore， this compound has exhibited
antinociceptive［18］，anticonvulsant［19］ and sedative activities［20］． Diisooctyl phthalate and dibutyl phthalate in
this essential oil are abundant． It may be related with environmental conditions，such as geographical
variations，climatic，soil composition and seasonal conditions．
Table 1 Chemical composition of essential oil of S． dulcificum
No． retention time /min component molecular formula GC content /%
1 3． 406 (Z)-2-heptenal C7H12O 0． 129
2 3． 595 hexanoic acid C6H12O2 0． 722
3 3． 665 phenol C6H6O 0． 487
4 3． 822 (E)-3-hexenoic acid C6H10O2 0． 857
5 4． 189 (E)-2-hexenoic acid C6H10O2 1． 292
6 4． 513 benzyl alcohol C7H8O 0． 477
7 4． 691 benzeneacetaldehyde C8H8O 0． 327
8 5． 469 guaiacol C7H8O2 0． 726
9 5． 610 linalool C10H18O 2． 139
10 5． 891 limonene C10H16 15． 805
11 5． 950 phenylethyl alcohol C8H10O 0． 197
12 6． 668 citronellal C10H18O 1． 187
13 6． 798 nonyl cyclopropane C12H24 0． 223
14 6． 852 citronellol C10H20O 0． 214
15 6． 955 4-ethylphenol C8H10O 1． 099
16 7． 468 (2E) ，6-dimethyl-3，7-octadiene-2，6-diol C10H18O2 0． 608
17 7． 565 α-terpineol C10H18O 1． 092
第 1 期 卢圣楼，等:神秘果叶挥发油化学成分分析及抗菌、抗肿瘤活性 125
continue to table 1
No． retention time /min component molecular formula GC content /%
18 8． 111 camphor C10H16O 1． 962
19 8． 332 nerol C10H18O 0． 477
20 8． 894 geraniol C10H18O 0． 809
21 9． 429 phloroglucitol C6H12O3 0． 204
22 9． 915 indole C8H7N 0． 837
23 10． 230 3，7-dimethyl-6-octenoic acid C10H18O2 0． 114
24 11． 249 geranic acid C10H16O2 0． 298
25 12． 292 tetradecane C14H30 0． 381
26 12． 405 vanillin C8H8O3 0． 200
27 12． 497 α-cedrene C15H24 0． 213
28 14． 696 pentadecane C15H32 0． 265
29 15． 133 2，4-bis(1，1-dimethylethyl)-phenol C14H22O 0． 382
30 17． 164 megastigmatrienone C13H18O 1． 412
31 17． 656 hexadecane C16H34 0． 391
32 17． 948 α-cedrol C15H26O 0． 214
33 18． 520 spathulenol C15H24O 24． 194
34 19． 752 triethyl citrate C12H20O7 0． 919
35 22． 939 tetradecanoic acid C14H28O2 0． 583
36 25． 246 5H-indeno［1，2-b］pyridine C12H9N 0． 251
37 29． 319 dibutyl phthalate C16H22O4 10． 326
38 29． 454 palmitic acid C16H32O2 4． 865
39 33． 835 phytol C20H40O 0． 203
40 34． 640 linolenic acid C18H30O2 0． 901
41 35． 353 octadecanoic acid C18H36O2 1． 276
42 44． 423 octadecane C18H38 0． 258
43 45． 665 diisooctyl phthalate C24H38O4 12． 402
44 46． 919 hexacosane C18H38 0． 221
2． 2 Antibacterial assay
The in vitro antibacterial potential of S． dulcificum essential oil against the employed bacteria was
quantitatively assessed by MIC values． Ｒesults on antibacterial activities involving the evaluation of inhibition
zones and MIC values are shown in Table 2． As it can be seen from Table 2，the result of the bioassay
exhibited that the essential oil displayed varying degrees of antibacterial activity against all tested bacterial
except P． aeruginosa． Additionally，the diameter of inhibition zone for the essential oil fell in the range of
3. 4 mm to 17． 5 mm． B． subtilis was the highest susceptibility with inhibition zone of (17． 50 ± 0． 8)mm，
and M． luteus appeared to be less susceptible with inhibition zone of (3． 43 ± 0． 6)mm． The minimum
inhibition concentration (MIC)value of this oil ranges between 39． 06 to 252． 15 mg /L． Among them，a
minimum of the essential oil was observed as the MIC (39． 06 mg /L)against B． subtilis，while the largest
MIC value of 252. 15 mg /L against M． luteus，respectively． Based on this finding，it can be concluded that
the Gram-positive bacterium is more susceptible to the antimicrobial properties of essential oils than Gram-
negative bacteria． It is considered to be due to its double layer cell membrane［21］．
The activity of the essential oil varies with its concentration and kind of bacteria． These differences in the
susceptibility of the test organisms to essential oil could be attributed to a variation in the rate of the essential
oil constituents penetration through the cell wall and cell membrane structures． The ability of essential oil to
disrupt the permeability barrier of cell membrane structures and the accompanying loss of chemiosmotic control
are the most likely reasons for its lethal action［22］． In addition，the components in lower amount may also
contribute to antibacterial activity of the essential oil，involving probably some type of synergism with other
active compounds． Ｒelated research on the essential oil of S． dulcificum is therefore important．
126 林 产 化 学 与 工 业 第 34 卷
Table 2 Antibacterial activity of leaf essential oil of S． dulcificum
microbial strain
diameter of inhibition zone /mm
essential oil ampicilin(contral)
MIC of essential oil /
(mg·L－1)
Bacillus subtilis 17． 50 ± 0． 8 26 ± 1． 1 39． 06
Staphylococcus aureus 6． 02 ± 0． 7 22 ± 1． 4 77． 23
Staphylococcus albus 5． 14 ± 1． 2 9 ± 0． 6 212． 31
Bacillus cereus 4． 21 ± 1． 0 12 ± 0． 7 200． 18
Micrococcus tetragenus 16． 92 ± 0． 7 15 ± 0． 5 97． 66
Micrococcus luteus 3． 43 ± 0． 6 28 ± 1． 2 252． 15
Escherichia coli 14． 60 ± 1． 0 20 ± 0． 8 78． 13
Pseudomonas aeruginosa — — —
2． 3 Antitumoral assay
To investigate the tumoricidal activity of S． dulcificum essential oil，four human cancer cell lines，i． e．，
SPC-A-1，BEL-7402，SGC-7901 and K562，were exposed to increasing concentrations of essential oils． Cell
viability was determined by the MTT assay． Table 3 presented the summarized inhibition rate (%)and half
inhibition concentration (IC50)． As shown in Table 3，it was found that the essential oil revealed different
cytotoxic activities towards the four human cancer cell lines investigated． In general，higher the concentration
of essential oil tested，the lower the cell viability percentages were． For the essential oil dilutions ranging from
1 mg /L to 100 mg /L，the essential oil induced a dose dependent inhibitory effect on K562，with IC50 values of
13． 5 mg /L． In comparison，at the higher dilution of 100 mg /L，the effect of the essential oil on the other
three cell lines was less sensitive．
Table 3 Cytotoxic activity and the IC50 of S． dulcificum essential oil against cell lines
cell lines
inhibition rate /%
essential oil 1 mg /L essential oil 10 mg /L essential oil 100 mg /L
IC50 /(mg·L－1)
SPC-A-1 — 9． 63 23． 74 ＞ 200
BEL-7402 — — 4． 62 ＞ 200
K562 23． 99 50． 81 67． 88 13． 5
SGC-7901 — 14． 36 27． 68 ＞ 200
The capacity to kill cancer cell lines was attributed to specific components of the oil． Previous
investigations have explained that monoterpenes and sesquiterpenes derivatives such as linalool，terpineol，
geraniol and selinene are reported to possess antibacterial，insect-repelling and toxic activities［23］． Since the
essential oil used in our study contained geraniol，α-terpineol and linalool(Table 1) ，we hypothesize that
these two constituents may be responsible for the observed antitumoral activity．
3 Conclusion
3． 1 The leaf essential oil of S． dulcificum was obtained by hydrodistillation． The chemical component of this
oil was investigated by GC-MS． Sixty-eight compositions were separated． Among them，44 components were
identified and represented 92． 14% of the total detected constituents． Spathulenol (24． 194%) ，limonene
(15． 805%) ，diisooctyl phthalate (12． 402%) ，dibutyl phthalate (10． 326%) ，palmitic acid (4． 865%)
and linalool (2． 139%)were the major constituents，and comprised 69． 731% of the essential oil．
3． 2 The essential oil displayed varying degrees of antibacterial activity against all tested bacterial except for
Pseudomonas aeruginosa，along with MIC values from 39． 06 to 252． 15 mg /L． The antitumoral activity using
MTT assay of the leaf essential oil indicated this oil was effective against human cancer cell line K562 in a
dose dependent manner，and its IC50 value was 13． 5 mg /L． A further study under in vitro condition is
recommended to elaborate the antibacterial and antitumoral activities of the major compounds of S． dulcificum
第 1 期 卢圣楼，等:神秘果叶挥发油化学成分分析及抗菌、抗肿瘤活性 127
essential oil for various applications．
Acknowledgment:The authors are thankful to Wang Jing，College of Chemistry and Chemical Engineering，Hainan Normal
University，China for her help in this research．
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