Pharmaceutical Biology, 2012; 50(11): 1397–1407

© 2012 Informa Healthcare USA, Inc.

ISSN 1388-0209 print/ISSN 1744-5116 online
DOI: 10.3109/13880209.2012.682116

Research Article

 nticancer activities of the extract from Longkong

A
(Lansium domesticum) young fruits
Aranya Manosroi1,2, Pensak Jantrawut1, Mathukorn Sainakham1, Worapaka Manosroi3, and
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Jiradej Manosroi1,2
1
Faculty of Pharmacy, 2Natural Products Research and Development (NPRDC), Science and Technology Research
Institute (STRI), Faculty of Pharmacy, and 3Faculty of Medicines, Chiang Mai University, Chiang Mai, Thailand

Abstract
Context: “Longkong”(Lansium domesticum Corr., Family: Meliaceae) is a fruit found in the south of Thailand. This plant
has been used in traditional medicines.
Objectives: To investigate the antiproliferative activities and the phytoconstituents of Longkong extracts.
Materials and methods: Cytotoxicity and apoptotic activity of 48 extracts were tested using the SRB assay and acridine
orange (AO)/ethidium bromide (EB) staining, respectively. The extracts which gave the highest anticancer activity
were selected to prepare the semipurified extracts and analysis for the constituents by gas chromatography–mass
For personal use only.

spectrometry (GC/MS).
Results: The highest percentage yield (59.38%) was from the cold water extract of Longkong ripe fruits (RFWC). The
highest total phenolic and flavonoid contents were observed in cold and hot methanol extract of Longkong stalks
(STMC and STMH). The hot and cold chloroform young Longkong fruit extracts (YFCH and YFCC) exhibited a cytotoxic
effect (IC50 < 1 mg/mL) against cancer cells. For apoptotic induction, YFCH demonstrated the highest activity against
KB of 13.84 ± 4.21% at 0.5 mg/mL which was 0.88 and 1.35 times of cisplatin and 5-FU, respectively, while apoptotic
cells in HT-29 were 8.68 ± 1.85% at 5 mg/mL, which was 0.61 and 1.43 times of cisplatin and 5-FU, respectively. YFCC
showed the highest apoptotic effect against KB cells at 10.70 ± 2.15% at 0.5 mg/mL, which was 0.68 and 1.07 times
of cisplatin and 5-FU, respectively. The major phytoconstituents in YFCH were hexadecanoic acid (11.53%) and ethyl
oleate (10.58%).
Discussion and conclusion: The crude extracts of Longkong showed anticancer activities and may provide new lead
compounds for the development of anticancer products.
Keywords:  Lansium domesticum, cytotoxicity, apoptotic activity, phytoconstituents, antiproliferative activity

Introduction
Cancer is characterized by uncontrolled cell proliferation
and differentiation. It can invade organs and tissues and
is the major public health burden both in developed and
developing countries. In 2002, it was estimated that there
were about 10.9 millions new cases, 6.7 million deaths,
and 24.6 million persons living with cancer around
the world (Parkin et al., 2005). The discovery of novel
anticancer agents from natural sources including plants,
marine organisms and micro-organisms was largely
based on the testing of cytotoxic activity against cancer
cell lines in vitro and animal cancer models in vivo.
Plants have a long history of being used in the treatment
of cancer. There are more than 3000 plant species that
have been reported for the treatment of cancer (Hartwell,
1982) such as vinblastine and vincristine, isolated from
the Madagascar Periwinkle, Catharanthus roseus G. Don.
(Apocynaceae) and Paclitaxel isolated from the bark
of Taxus brevifolia Nutt. (Taxaceae) (Cragg & Newman,
2005).

Address for Correspondence: Prof. Dr. Aranya Manosroi, PhD, Natural Products Research and Development Center (NPRDC), Science and
Technology Research Institute (STRI), Chiang Mai University, Chiang Mai 50200, Thailand. Tel.: 66-53-894806. Fax: 66-53-894169.
E-mail: pmpti005@chiangmai.ac.th
(Received 16 November 2011; revised 01 February 2012; accepted 29 March 2012)

1397
 1398  A. Manosroi et al.
Lansium domesticum Corr. (Meliaceae) is the popular
tropical plant producing economic edible fruits found
mainly in Southeast Asia, especially in the south of
Thailand. It is called “Longkong” in Thai. There are also
other cultivation of L. domesticum including Langsat (in
Thai), Duku-Langsat and Duku. According to the external
and internal characteristics of their fruits, Longkong is an
intermediate plant between Langsat and Duku-Langsat.
Longkong is almost seedless, thin skinned and free of
latex (Bamroongrugsa, 1992; Norlia, 1997). The edible
fruit portion of Longkong is 68% of the fruit weight. In 100
g, it contains 84 g of water, 14.2 g of carbohydrates, 0.8 g
of fiber, 0.6 g of ash, 19 mg of Ca, 275 mg of K, and some
protein, fat, vitamin B1, B2, and vitamin C. The fresh peel
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contains 0.2% of the light-yellow volatile oil, brown resin
and reducing acids. From the dried peel, it contains dark
semiliquid oleoresin composing of 0.17% volatile oil
and 22% resin (Heyne, 1987; Verheij & Coronel, 1992).
Five tetranorterpenoids, including domesticulide A-E,
were isolated from the seed of L. domesticum, together
with the 11 known triterpenoids. Six classes of limonoids
have been isolated from the seed extracts of L. domes-
ticum, including andirobin derivates, methyl angolen-
sates, mexicanolides, azadiradiones, onoceranoids, and
dukunolides (Tanaka et al., 2002). The peel, which is rich
in oleoresin, has been used against diarrhea and intes-
tinal spasms. Other parts of this plant have also been
For personal use only.
employed medicinally. The crushed seeds were used to
treat fevers. The astringent bark has been orally adminis-
tered against dysentery and malaria. The powdered bark
was used in poultices against scorpion stings. In Java, the
aromatic smoke from its burnt peel has served as a mos-
quito repellent and incense in the rooms of sick people
(Morton, 1987; Heyne, 1987; Verheij & Coronel, 1992).
However, limited information is available concerning the
anticancer activity of Longkong.
In this present study, the in vitro anticancer activities
in four cancer cell lines including antiproliferation and
apoptosis induction of the extracts from various parts of
Longkong harvested in the three provinces of Thailand
prepared by the six different processes were investigated
in order to evaluate their potential for cancer treatment.
Materials and methods
Materials
Acridine orange (AO), ethidium bromide (EB),
dimethyl sulfoxide (DMSO), sulphorodamine B, tris
(hydroxymethyl)-methlamine, trichloroacetic acid (TCA),
and Folin-Ciocalteu reagent were purchased from Sigma
Chemical Co. (St. Louis, MO). Sodium carbonate (Na2CO3)
and aluminium trichloride (AlCl3) were purchased from
Fluka (Buchs, Switzerland). Dulbecco’s modified Eagle’s
medium (DMEM), fetal bovine serum (FBS), penicillin and
streptomycin were obtained from GIBCO (Grand Island,
NY). All other analytical grade chemicals and reagents
were purchased from Wako Pure Chemical Industries, Ltd.
(Osaka, Japan).
 Pharmaceutical Biology
Plant selection
Longkong was collected from three provinces
(Narathiwat, Satun, and Yala) in the south of Thailand,
during October to November 2010 and separated into
eight parts including ripe fruits (RF), young fruits (YF),
old leaves (OL), young leaves (YL), seeds (SE), peels (PE),
stalk (ST), and branches (BR). The specimen was authen-
ticated by a botanist at the Faculty of Pharmacy, Chiang
Mai University in Thailand and deposited at NPRDC, at
Chiang Mai University in Thailand. (voucher specimen
no. NPRDC2010LK1-8). Descriptions and extract codes
of each Longkong samples including the plant parts from
each province are presented in Table 1.
Preparation of extracts
All Longkong parts were washed, cut into small pieces,
dried at 40 ± 2°C in a hot air oven, ground into powder
and kept in airtight plastic bags at 4 ± 2°C until use. For
the extraction process, the dried plant powder (100 g)
was extracted by mixing with solvents in six different
conditions. For HC (hot chloroform) and HM (hot meth-
anol), the powder was extracted by continuous Soxhlet
extraction for 1 h in 400 mL chloroform (60 ± 2°C) and
methanol (65 ± 2°C), respectively, until exhaustion. For
HW (hot water), the powder was boiled for 1 h with 400
mL distilled water at 100 ± 2°C and then cooled to room
temperature (25 ± 2°C). For CC (cold chloroform), CM
(cold methanol) and CW (cold water), 100 g of the pow-
der were macerated in 400 mL of chloroform, methanol
and distilled water, respectively and sonicated in a bath
sonicator for 1 h at room temperature (25 ± 2°C). The
mixtures were filtered through the Whatman No. 1 filter
paper and the plant residues were re-extracted twice
under the same conditions. The filtrate was pooled and
concentrated under vacuum by a rotary evaporator
(R-124 Buchi, Switzerland) and lyophilized. The dried
extracts were stored at 4 ± 2°C prior to use. Forty-eight
extracts were obtained and the percentage yields (w/w)
were calculated on the dry weight basis.
The total phenolic and flavonoid contents
The Folin-Ciocalteu method (Yeh & Yen, 2005) was used
to determine the total phenolic content of the extracts.
Briefly, 500 µL of the extract dissolved in 20% DMSO
at the concentration of 10 mg/mL were mixed with an
equal volume of 1 N Folin-Ciocalteu reagent and 1 mL
of 20% sodium carbonate (Na2CO3) and incubated for 2
h at room temperature (25 ± 2°C). The reaction mixture
was then centrifuged at 5000 rpm for 10 min. The
absorbance of the supernatant was measured at 730 nm
using a spectrophotometer. The experiments were done
in triplicate. The total phenolic contents were calculated
from the calibration curve of the standard gallic acid
[absorbance = 0.0009 gallic acid (µg) + 0.5089; R2 =
0.9891]. The mean of three readings was used and the
total phenolic content was expressed in mg of gallic acid
equivalents (GAE)/g of the extract.
 Antiproliferation effect of Longkong extract  1399

Table 1.  Comparison of the percentage yields of the 48 extracts The total flavonoid content was determined using
from eight plant parts of Longkong prepared by aqueous, the Dowd method (Arvouet-Grand et al., 1994). Briefly,
methanol and chloroform with cold and hot processes. 100 µL of 2% aluminium trichloride (AlCl3) in methanol
Extraction Extraction Extract were mixed with 100 µL of the extract (10 mg/mL) in 20%
Part used solvent temperature code % Yield
DMSO. Absorption readings at 415 nm by a microplate
Young fruits Chloroform Hot YFCH 9.29
reader (Biorad model 680, Biorad, Japan) were taken after
(Yala province) Cold YFCC 9.78 10 min against the blank sample consisted of 100 µL of
Methanol Hot YFMH 28.82 the extract solution mixed with 100 µL methanol without
Cold YFMC 38.7 AlCl3. The total flavonoid contents were calculated from
Water Hot YFWH 34.11 the calibration curve of the standard quercetin [absor-
Cold YFWC 38.4 bance = 7.5732 quercetin (µg) + 0.5742; R2 = 0.9526]. The
Ripe fruits Chloroform Hot RFCH 0.06 mean of three readings was used and the total flavonoid
(Narathiwat Cold RFCC 0.20 content was expressed in mg of quercetin equivalents
province)
Methanol Hot RFMH 32.12 (QE)/g of the extract.
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Cold RFMC 40.55

Water Hot RFWH 23.68 Cell culture
Cold RFWC 59.38 Human epidermal carcinoma (KB) which was obtained
Young leaves Chloroform Hot YLCH 5.71 from American Type Culture (ATCC), human colon
(Satun province) Cold YLCC 12.02 adenocarcinoma (HT-29) which was provided from
Methanol Hot YLMH 19.09 Medicinal Microbiology Department, Faculty of Biology,
Cold YLMC 30.64
University of Tuebingen, Tuebingen, Germany, hepato-
Water Hot YLWH 12.5
cellular carcinoma (HepG2) and murine melanoma cells
(B16F10) which were provided from Faculty of Tropical
Cold YLWC 21.05
Medicine, Mahidol University, Thailand were cultured
Old leaves Chloroform Hot OLCH 8.10
(Satun province) under the standard conditions in the complete culture
Cold OLCC 8.08
medium containing DMEM supplemented with 10%
Methanol Hot OLMH 14.58
(v/v) fetal bovine serum (FBS), penicillin (100 U/mL) and
For personal use only.

Cold OLMC 27.64

streptomycin (100 mg/mL). Cells were incubated in a
Water Hot OLWH 12.91
temperature-controlled, humidified incubator (Shel Lab,
Cold OLWC 19.71
model 2123TC, Cornelius, OR, USA) with 5% CO2 at 37°C.
Seeds Chloroform Hot SECH 1.31
(Narathiwat Cold SECC 0.67 Cytotoxicity test by the SRB assay
province)
Methanol Hot SEMH 44.61 Forty eight Longkong extracts and the standard anticancer
Cold SEMC 48.03 drugs (cisplatin and 5-FU), in concentrations ranging
Water Hot SEWH 1.03 from 0.1 to 1000 µg/mL, were tested for cytotoxic activity
Cold SEWC 0.53 in four cancer cell lines by the SRB assay (Papazisis et al.,
Peels Chloroform Hot PECH 11.65 1997). Briefly, the cells were plated at a density of 1.0 × 104
(Narathiwat Cold PECC 12.18 cells/well in 96-well plates and left for cell attachment on
province) the plate for 24 h in 5% CO2 at 37°C. Cells were then exposed
Methanol Hot PEMH 10.67
Cold PEMC 18.13 to five serial concentrations of the extracts (0.001–10 mg/
Water Hot PEWH 12.2 mL) for 24 h. After incubation, the cells were fixed with
Cold PEWC 17.65 50% trichloroacetic acid solution, incubated at 4°C for
Stalks Chloroform Hot STCH 17.88 1 h, and washed five times with distilled water. The excess
(Narathiwat Cold STCC 5.75 water was drained off and the plates were air-dried for 24
province)
Methanol Hot STMH 13.82
h. The cells were stained with 50 µL of 0.4% SRB solution in
Cold STMC 25.75
1% acetic acid for 30 min at room temperature (25 ± 2°C).
After incubation, the SRB solution was poured off and the
Water Hot STWH 15.25
plates were washed with 1% acetic acid until only the cell
Cold STWC 21.21
adhered dye was left. The plates were air-dried and 100
Branches (Satun Chloroform Hot BRCH 2.72
province) µL of 10 mM Tris-base solution were added to each well
Cold BRCC 2.71
to solubilize the dye. The mixture was shaken for 30 min
Methanol Hot BRMH 6.70
at room temperature (25 ± 2°C) and the absorbance was
Cold BRMC 8.36
measured at 540 nm by a microplate reader (Biorad model
Water Hot BRWH 11.96
680, Biorad, Japan). The experiment was performed
Cold BRWC 16.36
in triplicate. The percentages of cell viability were
BR, branches; CC, cold chloroform; CM, cold methanol; CW, cold calculated using the following equation: % cell viability
water; HC, hot chloroform; HM, hot methanol; HW, hot water; OL,
old leaves; PE, peels; RF, Ripe fruits; SE, seeds; ST, stalk; YF, young = (Asample − Ablank/Acontrol − Ablank) × 100, where Asample was
fruits; YL, young leaves. the optical density of the cells treated with the samples,

© 2012 Informa Healthcare USA, Inc.

 1400  A. Manosroi et al.
Acontrol was the optical density of the nontreated cells, and
Ablank was the optical density of the Tris-base solution
at time zero. The IC50 values (the concentrations that
inhibit 50% of cell viability) were obtained by plotting the
percentages of cell viability versus the concentrations of
the samples.
Apoptotic activity by AO and EB double staining
From the SRB assay, the extracts which gave the IC50 value
of less than 1000 μg/mL were selected to investigate
for apoptotic activity in four cancer cell lines by AO/EB
staining. After incubation, the cells were treated with the
extracts at the three final concentrations of 0.05, 0.5 and
5 mg/mL and incubated for 24 h. To stain the apoptotic
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cells, 10 μL of the AO/EB dye mix (100 μg/mL of AO and
100 μg/mL of EB in PBS) were added to each well and the
apoptotic and necrotic cells were viewed and counted
under a fluorescent microscope with the total minimum
of 100 cells (Ribble et al., 2005). The experiments were
done in triplicate.
The solvent partition of the selected Longkong
extracts
The Longkong extract which showed the highest cyto-
toxicity by the SRB assay and the apoptotic by AO/EB
staining was selected to solvent partitioning to prepare
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the semipurified fractions. The selected chloroform
extract (1.0 g) was partitioned between 100 mL of H2O
and 100 mL of ethyl acetate, leading to the water frac-
tion. The residue was partitioned between 200 mL of
53% methanol and hexane to give the methanol and
hexane fractions. The compounds in these fractions
were identified by gas chromatography–mass spec-
trometry (GC/MS) analysis.
GC/MS analysis
The fractions of Longkong crude extracts which showed
the highest cytotoxicity by SRB assay and apoptotic activ-
ity by AO/EB were analyzed for the compounds by GC/
MS according to the method of Instrumentation and
Chromatographic System Gas Chromatography-Mass
Spectrometry Model Agilent (Agilent Hewlett Packard,
Waldbronn, Germany). An Agilent HP-5MS capillary
column (30 m × 0.32 mm i.d., film thickness 0.25 μm;
Agilent Hewlett Packard, Waldbronn, Germany) and a
split/splitless injector was used for the separation. The
1 μL sample was injected into the injector with the split
ratio of 1:50. The oven temperature was kept at 50°C for
1 min, then increased to 180°C at the rate of 8°C/min for
1 min and finally increased to 280°C at the rate of 5°C/min
for 3 min. The temperature of the injector, detector and
ion source was 250°C. Helium was used as the carrier gas.
Identification of the compounds was based on the com-
parisons of their mass spectra with those recorded in the
National Institute of Standards and Technology (NIST)
database.
 Pharmaceutical Biology
Statistical analysis
The results are presented as the mean of three indepen
dent experiments and analyzed by SPSS (version 16.0,
SPSS Inc., IL, USA). ANOVA was used for the analysis of
the test results (LSD test) at the significance level of p value
<0.05. Correlation coefficients (r) were used to determine
the relationship between the variables which were calcu-
lated by the bivariate correlation statistical function.
Results and discussion
Percentage yields of Longkong extracts
prepared by the six extraction processes
The percentage yields of the 48 extracts prepared by
six extraction processes (CC, CH, MC, MH, WC, and
WH) by various solvents, temperatures and parts used
of Longkong are summarized in Tables 1 and 3. The
methanol and aqueous extracts showed summation
yields which were calculated by the summation of the
percentage extract yields of each solvent (408.21 and
317.93%, respectively) significantly higher (p < 0.05) than
the chloroform extracts (summation yields of 108.05%).
This indicated that Longkong may contain the polar
more than the nonpolar compounds. In all solvent sys-
tems, the cold (room temperature; 25 ± 2°C) process
(summation yields of 483.48%) gave significantly higher
(p < 0.05) yields than the hot (60–100°C) process (sum-
mation yields of 350.71%). The heat labile components
in Longkong may be degraded at high temperature. For
the parts of Longkong, the young and ripe fruits (sum-
mation of percentage yields at 159.10 and 155.93, respec-
tively) gave higher yields than other plant parts. The high
yields of the fruit extracts may be from the carbohydrates
which were the constituents mainly found in the fruit of
L. domesticum (Morton, 1987). The highest percentage
yield was from the cold water extract of the ripe fruits
(RFWC) at 59.38%, whereas the lowest percentage yield
was from the hot chloroform extract of the ripe fruits
(RFCH) at 0.06%.
Total phenolic and flavonoid contents
Table 2 showed the total phenolic and flavonoid contents
of the 48 extracts from eight plant parts of Longkong. The
total phenolic contents (µg of GAE/g ± SD) varied from
0 to 272.89, whereas the total flavonoid contents (µg
of QE/g ± SD) varied from 0 to 57.88. The highest total
phenolic and flavonoid contents were observed in cold
and hot methanol stalk extracts (STMC and STMH) with
the GAE and QE values of 272.89 ± 3.93 and 57.88 ± 5.18
µg, respectively. All chloroform extracts gave the total
phenolic and flavonoid contents at the summation of
GAE and QE at 90.04 and 0 µg, respectively which were
significant lower (p < 0.05) than the methanol extracts
(summation of GAE and QE at 1203.75 and 192.16 µg,
respectively) and water extracts (summation of GAE
and QE at 1113.52 and 189.45 µg). For the extraction
temperatures, the extracts by the cold process gave
 Antiproliferation effect of Longkong extract  1401

Table 2.  Total phenolic and flavonoid contents and cytotoxic activity (IC50) on four cancer cell lines of the 48 extracts from eight plant parts
of Longkong and standard anticancer drugs.
Total phenolic content Total flavonoid content Cytotoxicity IC50 (µg/mL)
Extract code (µg GAE/g ± SD) (µg QE/g ± SD) KB HT-29 HepG2 B16F10
YFWH 0.03 ± 0.10 ND – – – –
YFWC 0.01 ± 0.01 ND – – – –
YFMH 4.00 ± 0.47 ND – – – –
YFMC 2.53 ± 0.06 ND – – – –
YFCH ND ND – 955.64 ± 25.25 934.00 ± 46.20 421.5 ± 12.98
YFCC ND ND 983.81 ± 13.17 934.89 ± 52.43 – 712.28 ± 2.69
RFWH 86.78 ± 27.50 ND – – – –
RFWC 233.44 ± 18.86 ND – – – –
RFMH 105.67 ± 9.67 ND – – – –
RFMC 120.67 ± 5.01 ND – – – –
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RFCH ND ND – – – –
RFCC ND ND – – – –
YLWH 12.08 ± 0.10 1.18 ± 0.10 – – – –
YLWC 12.39 ± 0.25 0.77 ± 0.02 – – – –
YLMH 8.07 ± 0.20 0.02 ± 0.00 – – – –
YLMC 0.09 ± 0.00 ND – – – –
YLCH ND ND – – – –
YLCC ND ND – – – –
OLWH 15.91 ± 0.36 ND – – – –
OLWC 14.83 ± 0.37 ND – – – –
OLMH 13.92 ± 0.56 ND – – – –
OLMC 12.36 ± 0.38 ND – – – –
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OLCH 0.96 ± 0.08 ND – – – 734.01 ± 40.38

OLCC 0.66 ± 0.45 ND – – – –
SEWH 21.59 ± 0.00 49.11 ± 1.70 – – – –
SEWC 108.44 ± 0.79 39.09 ± 6.65 – – – –
SEMH 61.96 ± 18.86 4.51 ± 2.27 – – – –
SEMC 147.89 ± 3.14 3.38 ± 4.19 – – – –
SECH 17.89 ± 6.78 ND – – – –
SECC 16.41 ± 2.36 ND – – – –
PEWH 50.77 ± 8.67 35.67 ± 9.43 – – – –
PEWC 49.86 ± 8.44 45.11 ± 3.44 – – – –
PEMH 60.11 ± 1.57 33.11 ± 2.00 – – – –
PEMC 240.11 ± 17.64 44.45 ± 2.82 – – – –
PECH 20.67 ± 8.43 ND – – – –
PECC 33.45 ± 49.86 ND – – – –
STWH 248.44 ± 3.93 10.83 ± 6.11 – – – –
STWC 257.89 ± 12.57 8.87 ± 1.52 – – – –
STMH 140.85 ± 25.93 57.88 ± 5.18 – – – –
STMC 272.89 ± 3.93 48.59 ± 4.13 – 665.34 ± 92.47 510.14 ± 14.84 –
STCH ND ND – – – –
STCC ND ND – 906.19 ± 34.30 – 638.73 ± 59.32
BRWH 1.06 ± 0.04 ND – – – –
BRWC ND ND – – – –
BRMH 6.37 ± 0.05 0.11 ± 0.03 – – – –
BRMC 6.26 ± 0.19 0.11 ± 0.03 – – – –
BRCH ND ND – – – –
BRCC ND ND – – – –
Standard anticancer drug
 Cisplatin 12.72 ± 2.34 0.04 ± 0.01 141.73 ± 11.55 57.85 ± 9.77
 5-FU 12.94 ± 5.67 155.18 ± 21.76 203.18 ± 19.89 14.76 ± 2.24
–, IC50 values were more than 1000 µg/mL; BR, branches; CC, cold chloroform; CM, cold methanol; CW, cold water; HC, hot chloroform;
HM, hot methanol; HW, hot water; ND, the contents can not be detected; OL, old leaves; PE, peels; RF, Ripe fruits; SE, seeds; ST, stalk; YF,
young fruits; YL, young leaves.

© 2012 Informa Healthcare USA, Inc.

 1402  A. Manosroi et al.
significant higher (p < 0.05) total phenolic contents
(summation of GAE at 1530.18) than that by the hot
process (summation of GAE at 877.13). However, there
was no difference in the total flavonoid contents between
the cold and hot extracts. The high temperature appeared
not to destroy the flavonoid aglycone which agreed with
the previous report (Rossi et al., 1986). However, the
degradation of some compounds in Longkong including
phenolics may be accelerated by high temperature (Burg
& Fraile, 1995; Tomaino et al., 2005), thereby changing
of their structures and activities. The stalk and peel
extracts exhibited higher total phenolic and flavonoid
contents with the summation of GAE and QE at 920.07
and 158.34, respectively (Table 3). Phenolics, such as
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methanol and chloroform extracts of stalks (STMC and
STCC) exhibited cytotoxic activity. The YFCH, YFCC,
OLCH, and STCC extracts demonstrated cytotoxic
effect against B16F10 cancer cell line with IC50 values of
421.5 ± 12.98, 712.28 ± 2.69, 734.01 ± 40.38, and 638.73
± 59.32 µg/mL, respectively. The YFCH and STMC
extracts showed a cytotoxic effect against HepG2 with
IC50 values of 934.00 ± 46.20 and 510.14 ± 14.84 µg/mL,
respectively. For the HT-29 cancer cell line, YFCH, YFCC,
STMC, and STCC extracts gave a cytotoxic effect with IC50
values of 955.64 ± 25.25, 934.89 ± 52.43, 665.34 ± 92.47,
and 906.19 ± 34.30 µg/mL, respectively, whereas only
YFCC exhibited a cytotoxic effect against the KB cancer
cell line with an IC50 value of 983.81 ± 13.17 µg/mL. The

polyphenols and flavonoids, which have at least one
aromatic ring bearing one or more hydroxyl groups,
are found in glycolated form in plant cells. Infact, the
decrease of degradation, reduction of toxic effects and
the facilitation of the transport across the membranes
of the phenolics can be modified by increasing their
water solubility (Jones & Vogt, 2001). Naturally, these
compounds are located in the vacuoles within the plant
cells and are in the polar soluble fraction. They can be
therefore easily extracted with the polar solvent such as
water and methanol (Rispail et al., 2005).
Cytotoxic activity on cancer cell lines
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young fruit extracts including YFCH and YFCC showed
a cytotoxic effect against all cancer cell lines. Most chlo-
roform extracts showed cytotoxic effects. On the B16F10
cell line, the YFCH extract gave a potency of about 0.14
and 0.04 times of cisplatin and fluorouracil, respectively.
It appeared that there was no correlation between the
cytotoxic activity and the total phenolic and flavonoid
content (R = −0.1872 to 0.1174). This may indicate that
the phenolic and flavonoid compounds contained in the
extracts are not related to the cytotoxic activity, but the
nonpolar compounds contained especially in the chlo-
roform extracts may be related to the cytotoxic activity.
Triterpenoids which are the major nonpolar second-

The cytotoxicity (IC50 values) of the 48 extracts from eight ary metabolite constituents in L. domesticum may be
parts of Longkong and the standard anticancer drugs responsible for the cytotoxic effects since the significant
on four cancer cell lines are shown in Table 2. Only inhibitory activity of skin-tumor promotion on Epstein-
five extracts (10% of 48 extracts) from three plant parts Barr virus activation of these compounds have been
(37.5% of eight plant parts) including the cold and hot reported (Nishizawa et al., 1989). In fact, there were few
chloroform extracts of young fruits (YFCC and YFCH), reports on the effects of L. domesticum extracts or their
the hot chloroform extract of old leaves (OLCH), the cold isolated compounds on cancer cell lines. The hot and
cold chloroform extracts of the young fruits of Longkong
which gave the highest cytotoxic activity were selected
Table 3.  Summations of the percentage yields, total phenolic, for further apoptosis induction assays.
and flavonoid contents depending on the extraction solvent,
temperature, and part used of the 48 Longkong extracts.
Summation of
Apoptotic assay
% Yield GAE QE
The necrotic and apoptotic cells of the four cancer cell
Extraction solvent
lines were not observed in the negative control group.
 Chloroform 108.05 90.04 0 The percentages of apoptotic and necrotic cells of the
 Methanol 408.21 1203.75 192.16 cold and hot chloroform extracts of young fruits (YFCC
 Water 317.93 1113.52 189.45 and YFCH) on four cancer cell lines are shown in Table
Extraction temperature 4. The morphological features of the earliest apoptosis
 Hot 350.71 877.13 191.24 of KB cells treated with the YFCC and YFCH extracts
 Cold 483.48 1530.18 190.37 and the apoptotic cells of HT-29 cells treated with the
Longkong part used YFCH extract are shown in Figure 1. However, no obvious
  Young fruits 159.10 6.57 0 apoptotic morphological changes were observed in the
  Ripe fruits 155.93 546.56 0 treated samples in HepG2 and B16F10 cell lines.
  Young leaves 101.01 32.63 0.79 For apoptotic induction, the standard anticancer
  Old leaves 91.02 58.64 0 drugs (cisplatin and 5-FU) at 0.05 mg/mL induced the
 Seeds 96.18 374.18 96.09 activity in all cancer cell lines, but the YFCH extract
 Peels 82.48 454.97 158.34 showed no effects at this concentration. The YFCH
 Stalks 99.66 920.07 126.17 extract indicated the highest apoptotic cells of 13.84 ±
 Branches 48.81 13.69 0.22 4.21% at 0.5 mg/mL and 8.68 ± 1.85% at 5 mg/mL against
GAE, mg of gallic acid equivalents/g of the extract; QE, mg of KB and HT-29 cell lines, respectively. YFCC also gave
quercetin equivalents/g of the extract. the highest apoptotic effect against KB of 10.7 ± 2.15% at

 Pharmaceutical Biology
 Antiproliferation effect of Longkong extract  1403

Table 4.  The percentages of the apoptotic and necrotic cell numbers in four cancer cells induced by the hot and cold chloroform extracts of
the young Longkong fruits (YFCH and YFCC).
Conc. KB HT-29 HepG2 B16F10
Sample (mg/mL) % A ± SD % N ± SD % A ± SD % N ± SD % A ± SD % N ± SD % A ± SD % N ± SD
YFCH 0.05 – – – – – – – –
0.5 13.84 ± 4.21 – 5.95 ± 1.85 – – – – –
5 12.12 ± 1.01 6.19 ± 2.93 8.68 ± 1.85 – – – – 27.58 ± 6.66
YFCC 0.05 – – – – – – – –
0.5 10.70 ± 2.15 – – – – – – 81.02 ± 8.37
5 2.00 ± 1.06 45.36 ± 13.71 – 41.13 ± 6.44 – 100 ± 0.00 – 100 ± 0.00
Cisplatin 0.05 15.80 ± 6.17 1.36 ± 1.24 14.25 ± 2.54 – 25.46 ± 4.49 2.76 ± 1.55 10.31 ± 2.46 –
5-FU 0.05 10.22 ± 4.42 – 6.09 ± 2.98 – 3.53 ± 2.20 – 4.67 ± 1.52 –
–, the percentage of the cells cannot be detected; A, apoptotic cell numbers; N, necrosis cell numbers; YFCC, Longkong young fruit crude
extract prepared by cold chloroform extraction; YFCH, Longkong young fruit crude extract prepared by hot chloroform extraction.
Pharmaceutical Biology Downloaded from informahealthcare.com by University of British Columbia on 12/10/14
For personal use only.

Figure 1.  Apoptotic morphology by AO/EB staining in cancer cells treated with the hot (YFCH) and cold (YFCC) chloroform extracts of the
Longkong young fruits observed by fluorescent light (left) with the excitation and emission wavelength of AO and EB at 502 and 510; 526 and
595 nm, respectively, and light microscope (right). Cell apoptosis was caused by YFCH at 0.5 mg/mL in the KB cell line (a, b), YFCH at 5 mg/
mL in HT-29 cell line (c, d), and YFCC at 0.5 mg/mL in KB cell line (e, f ). White arrows indicated apoptotic cells.

0.5 mg/mL. Triterpenoids in L. domesticum may be the
major constituents that are responsible for this effect,
since they have been reported to inhibit tumor initiation
and promotion (Oguro et al., 1998) and induce apoptosis
(Choi et al., 2000; Lauthier et al., 2000). YFCH showed the
highest necrotic induction of 6.19 ± 2.93% at 5 mg/mL,
and 27.58 ± 6.66% at 5 mg/mL in KB and B16F10 cell lines,
respectively. In all cancer cell lines, YFCC gave the high-
est necrotic cells of 45.36 ± 13.71% at 5 mg/mL, 41.13 ±
6.44% at 5 mg/mL, 100 ± 0% at 5 mg/mL, and 100 ± 0%
at 5 mg/mL in KB, HT-29, HepG2, and B16F10 cell lines,
respectively. The necrotic induction increased with the

© 2012 Informa Healthcare USA, Inc.

 1404  A. Manosroi et al.
increase in concentration of the extracts. At high con-
centrations, the inhibition of the cell cycle progression
can result in cell death by necrotic induction (Yeung et
al., 1999). This result has indicated that the extract from
the hot chloroform extracts of the young Longkong fruits
(YFCH) showed potent apoptotic and necrotic induction
in all cancer cell lines, which can be further developed as
an anticancer drug.
GC/MS analysis
The YFCH which demonstrated promising cytotoxic
and apoptotic induction activity was selected to
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For personal use only.
prepare semipurified fractions. The separation
chromatograms of YFCH crude extract and their
fractions including hexane (YFCHhex), methanol
(YFCHmet) and water (YFCHwat) fractions were run in 40
min and presented in Figure 2. The components were
identified by mass spectroscopy and their retention
times were compared with the NIST database. The
total numbers of the components identified in the
YFCH crude extract and their three fractions including
(YFCHhex, YFCHmet, and YFCHwat) were 48, 39, 16, and
14 compounds, respectively. The major components
which gave the percentages of correlation maximum

Figure 2.  GC/MS chromatograms of YFCH crude extract (Longkong young fruit prepared by hot chloroform extraction) and their fractions
(hexane, methanol, and water fractions).

 Pharmaceutical Biology
 Antiproliferation effect of Longkong extract  1405

Table 5.  GCMS analysis of the hot chloroform crude extracts (YFCH) and their fractions of Longkong young fruits by solvent–solvent
partitioning method.
GCMS RT Compound % Corr. max Total (%)
YFCH crude extract
 14.200 α-cubebene 56.72 5.971
 15.836 1,2,4a,5,6,8a-hexahydro-4,7-dimethyl-1-(1-methylethyl)-naphthalene 64.78 6.856
 16.151 α-muurolene 16.53 1.750
1,2,4a,5,6,8a-hexahydro-4,7-dimethyl-1-(1-methylethyl)-(1α,4aα,8aα)-naphthalene
 16.426 1,2,3,4,4a,5,6,8a-octahydro-7-methyl-4-methylene-1-(1-methylethyl)-(1α,4aβ,8aα)- 46.77 4.950
naphthalene
 16.517 1,2,3,4-tetrahydro-1,1,6-trimethyl-naphthalene 21.03 2.226
 17.433 (−)-spathulenol 26.25 2.778
 18.137 1,2,3,4-tetrahydro-3-isopropyl-5-methyl-1-oxonaphthalene 31.48 3.332
 18.549 α-cadinol 43.72 4.642
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(+)-cycloisosativene
 19.006 ledene oxide-(II) 26.16 2.769
6-isopropenyl-4,8a-dimethyl-1,2,3,5,6,7,8,8a-octahydro-naphthalenol
 21.078 aristolene epoxide 25.00 2.646
2,5-bis(1,1-dimethylethyl)-phenol
 22.806 hexadecanoic acid 74.06 11.531
 24.860 ethyl oleate 100.00 10.584
 25.037 octadecanoic acid 57.16 6.050
 37.774 17-(1,5-dimethylhexyl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H- 4.624 4.624
cyclopenta [a]phenanthrenol
Hexane fraction from YFCH crude extract
 14.223 α-cubebene 100.00 14.982
 15.831 1,2,4a,5,6,8a-hexahydro-4,7-dimethyl-1-(1-methylethyl)-naphthalene 75.18 11.263
For personal use only.

 16.414 1,2,3,4,4a,5,6,8a-octahydro-7-methyl-4-methylene-1-(1-methylethyl)-(1α,4aβ,8aα)- 46.92 7.030

naphthalene
 16.529 1S-cis-1,2,3,5,6,8a-hexahydro-4,7-dimethyl-1-(1-methylethyl)-naphthalene 50.64 7.587
 17.410 (−)-spathulenol 23.81 3.567
 18.520 α-cadinol 27.90 4.181
(+)-cycloisosativene
 18.989 ledene oxide-(II) 20.63 3.090
6-isopropenyl-4,8a-dimethyl-1,2,3,5,6,7,8,8a-octahydro-naphthalenol
 20.814 1,5,5-trimethyl-6-acetylmethyl-cyclohexene 20.43 3.062
Methanol fraction from YFCH crude extract
 22.846 hexadecanoic acid 56.50 28.359
 24.928 octadecanoic acid 100.00 50.195
Water fraction from YFCH crude extract
 36.910 8S-cis-2,4,6,7,8,8a-hexahydro-3,8-dimethyl-4-(1-methylethy lidene)-5(1H)-azulenone 100.00 29.287
3,5,6,7,8,8a-hexahydro-4,8a-dimethyl-6-(1-methylethenyl)-2(1H)-naphthalenone
 38.123 1,2,3,3a,8,8a-hexahydro-5-methoxy-3a,8-dimethy pyrrolo[2,3-b]indole 87.53 25.636
6-ethyl-2,5-dihydroxy1,4-naphthoquinone
The major peaks which gave the percentages of correlation maximum area (% corr. max) more than 15 were selected to present.
YFCH, Longkong young fruit crude extract prepared by hot chloroform extraction.

area (% corr. max) of more than 15 are presented in
Table 5. The major phytoconstituents in the fresh
Longkong young fruit crude extract by hot chloroform
(YFCH) were hexadecanoic acid (11.53%), ethyl oleate
(10.58%), 1,2,4a,5,6,8a-hexahydro-4,7-dimethyl-1-
(1-methylethyl)-naphthalene (6.86%), octadecanoic
acid (6.05%) and α-cubebene (5.97%). Some major
phytoconstituents in all fractions were similar to
the crude extract, but had higher amount than the
YFCH crude extract. The YFCHmet fraction gave 28.36
and 50.20% of hexadecanoic and octadecanoic acids
which were 2.46 and 8.30 times that of the YFCH crude
extracts, respectively. The YFCHhex fraction gave 14.98%
of α-cubebene which was 2.51 times that of the YFCH
crude extracts. GC/MS of the Longkong crude extracts
also contained two fatty acids including hexadecanoic
and octadecanoic acid as the major components but
at lower amounts of 11.53 and 6.05%, respectively.
Therefore, the anticancer effect of YFCH might be
from these fatty acids. It has been reported that high
concentrations of certain fatty acids can cause cell
death via apoptosis or necrosis (Andrade et al., 2005).

© 2012 Informa Healthcare USA, Inc.

 1406  A. Manosroi et al.
Palmitic acid (hexadecanoic acid) has been reported to
induce apoptosis in tumor cells (Kong & Rabkin, 2002;
Harada et al., 2002). Previous reports have mentioned
that naphthalene derivatives have demonstrated strong
growth inhibitory activity against cell lines (Robert
et al., 1961; Wang et al., 1998). However, not only these
fatty acids might be responsible for the anticancer
activity, but also other compounds, including the
naphthalene derivatives, the intermediate products of
resin production, α-cubebene and the sesquiterpene
volatile oil as well. In addition, the crude extracts
of Longkong which contained these compounds
demonstrated more anticancer activity than their
fractions owing to their synergistic effects with the
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fatty acids (Hamburger & Hostettmann, 1991; Karagöz
et al., 2007).
Conclusion
The present study has demonstrated the in vitro antican-
cer activities of Longkong extracts cultivated in Thailand.
About 10% of the 48 extracts of eight parts and from three
provinces in Thailand of Longkong showed antiprolifera-
tive activity in four cancer cell lines. The extract of Longkong
young fruit by hot and cold chloroform extraction (YFCH
and YFCC) showed a higher cytotoxic effect than other
extracts against all cancer cell lines. The nonpolar com-
For personal use only.
pounds containing in the extracts may be responsible for
the cytotoxic activity. The extract of Longkong young fruit
by hot chloroform (YFCH) exhibited the most potent cyto-
toxic activity in B16F10 cell line with an IC50 of 421.50 ± 12.98
which was 7.29- and 28.56- times less potent than cisplatin
and 5-FU, respectively. This extract also showed apoptotic
activity with the KB cell line of 13.84 ± 4.21% which was
0.88- and 1.35- times of cisplatin and 5-FU, respectively,
while apoptotic cells in HT-29 were 8.68 ± 1.85%, which was
0.61- and 1.43- times of cisplatin and 5-FU, respectively.
The phytochemical contents of the YFCH crude extract and
its fractions from hexane, methanol and water fractions
were identified by GC/MS analysis. The major constituents
in all three fractions and the crude extract were the same
which were hexadecanoic acid, octadecanoic acid, and
α-cubebene, but with higher amount in the fractions than
in the crude extract. The results from this study have dem-
onstrated that the anticancer activity of the hot chloroform
extract from the young fruits of Longkong might be from
these compounds which can be further developed for the
treatment of oral and colorectal cancers.
Declaration of interest
This work was supported by the Agricultural Research
Development Agency (Public Organization) the invest-
ment funds following the Royal Decree (ARDA) in Thailand
and Natural Products Research and Development Center
(NPRDC), Science and Technology Research Institute
(STRI), Chiang Mai University, Thailand.
 Pharmaceutical Biology
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