Future Journal of Pharmaceutical Sciences xxx (2017) 1e4

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Future Journal of Pharmaceutical Sciences

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pharmaceutical-sciences/

Bioassay guided fractionation and cytotoxic activity of Daucus carota

var. boissieri
Noha Khalil a, *, Mohamed Ashour b, Abdel Naser Singab b, Osama Salama a
a
Department of Pharmacognosy, Faculty of Pharmaceutical Sciences and Pharmaceutical Industries, Future University in Egypt, 11835, Fifth Settlement,
Cairo, Egypt
b
Department of Pharmacognosy, Faculty of Pharmacy, Ain Shams University, Abbasia, 11566, Cairo, Egypt

a r t i c l e i n f o a b s t r a c t

Article history: The hexane extract and the hydro-distilled essential oil from red carrot fruits (Daucus carota var. boissieri)
Received 6 February 2017 were evaluated for their cytotoxic activity against human tumor breast cell lines (MCF-7). Cell viability
Received in revised form was evaluated by MTT assay. The extract exhibited good cytotoxic activity shown through its low IC50
14 June 2017
(9.12 ± 0.58 mg/ml) against the standard 5-Flououracil (8.46 ± 0.63 mg/ml). Phytochemical investigation
Accepted 10 July 2017
Available online xxx
of the hexane extract using column chromatography yielded three compounds; 8-methoxypsoralen (1),
a-asarone (2) and 3,4,5-trimethoxy-benzaldehyde (3), a compound isolated for the first time from
D. carota and from family Apiaceae. Structure elucidation of the isolated compounds was carried out on
Keywords:
Apiaceae
the basis of their spectral data analysis (IR, MS, 1H NMR an 13C NMR) The three isolated compounds were
Red carrot evaluated for their cytotoxic activity using the same conditions. Only compound (1) exhibited good
Hexane cytotoxic activity (IC50; 9.38 ± 0.78 mg/ml), compound (2) had moderate activity (46.12 ± 1.31 mg/ml),
Cytotoxicity while compound (3) had no cytotoxic activity (100.6 ± 3.11 mg/ml). These compounds need to be more
MCF-7 investigated against other cell lines; also they are considered as a good substrate for future SAR study and
modifications to produce more potent cytotoxic derivatives.
© 2017 Future University. Production and hosting by Elsevier B.V. This is an open access article under the
CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

1. Introduction showed antioxidative and anticancer effects [8].

Daucus L., a member of family Apiaceae comprises around 60
species spread over Africa, Europe and West Asia. Six wild Daucus
species are present in Egypt [1]. Daucus carota var. boissieri (red
carrot) and D. carota var. sativus (yellow carrot) are widely culti-
vated for their edible roots [2]. D. carota has been reported to
contain several classes of phytoconstituents as essential oils, phe-
nylpropanoids, polycetylenes as well as flavonoids [3]. Cultivated
carrot is mainly used as a root vegetable, while its fruit essential oil
is sometimes used as a flavouring agent in food products and in
cosmetics [4]. The wild form is reported to have medicinal value
since ancient times [5]. Carrot fruit extracts are reported to have
anti-nociceptive and anti-inflammatory effects [6], as well as
hypoglycaemic and antidiabetic activity [7]. Also the fruit extracts
Cancer remains a leading cause of death worldwide, and the
growing interest in natural products with anticancer activities has
led to the discovery of many promising compounds, among which
plant secondary metabolites such as terpenes, phenolics and al-
kaloids [9,10]. Monoterpenes, sesquiterpenes and phenyl-
propanoids of essential oil derived from different medicinal plants
exhibit antioxidant and anticancer properties [11,12]. Phenolics and
flavonoids from various plant foods are also known to possess
potent antioxidant activity and cytotoxicity against a variety of
human cancer cell lines [13,14].
The present study aims at the phytochemical and cytotoxic
study of the hexane extract of the D. carota var. boissieri fruits with
the isolation and structure elucidation of compounds which may
have promising cytotoxic activity.

2. Material and methods

* Corresponding author.
E-mail addresses: noha.hassan@fue.edu.eg (N. Khalil), ml_ashour@hotmail.com
(M. Ashour), nasersingab@hotmail.com (A.N. Singab), osalama99@hotmail.com
2.1. Preparation of the hexane extract
(O. Salama).
Peer review under responsibility of Future University. The fruits were collected from a cultivated field at El-Sharkeyya

http://dx.doi.org/10.1016/j.fjps.2017.07.002
2314-7245/© 2017 Future University. Production and hosting by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/
licenses/by-nc-nd/4.0/).

Please cite this article in press as: N. Khalil, et al., Bioassay guided fractionation and cytotoxic activity of Daucus carota var. boissieri, Future
Journal of Pharmaceutical Sciences (2017), http://dx.doi.org/10.1016/j.fjps.2017.07.002
2 N. Khalil et al. / Future Journal of Pharmaceutical Sciences xxx (2017) 1e4
governorate, Nile delta of Egypt during summer 2010. Plants were
identified and authenticated according to Boulos [1] and identified
by Prof. Abdel Sallam Al-Newaihi, Department of Botany, Faculty of
Science, Ain Shams University, Egypt. The hexane extract of Daucus
carota var. boissieri was prepared by solvent extraction with double
distilled hexane (B.P. 40e60
C). Three litres of the solvent were
added to two kg of the fruits and macerated for 3 days and filtered
after that. The process was repeated till exhaustion. The solvent
was distilled off using rotary evaporator under reduced pressure.
2.2. Column chromatography
Twenty grams of the n-hexane fraction was dissolved in mini-
mum amount of hexane and applied on top of 600 g silica gel 60
column chromatography (120  4 cm). The column was eluted with
n-hexane with gradual increase of ethyl acetate. A minimum of 50
fractions of 250 ml each, were collected for each solvent system and
monitored by TLC on silica gel 254 sheets. Chromatograms were
sprayed with 10% sulphuric acid and heated to 100
C in an oven for
seven minutes
2.3. Structural elucidation experiments
IR spectra (KBr) were recorded on a Perkin Elmer FTIRspec-
trometer. MS were measured on a GSMSQP1000EX gas chroma-
tographmass spectrometer Shimadzu (Japan). For column
chromatography, silica gel (Merck, 63200 mm particle size) was
used. For NMR experiments, samples were dissolved in 0.75 ml of
deuterated solvents and measured in 5 mm tubes at 25
C on either
Varian 300 MHz Mercury plus NMR spectrometer or Varian A
300 MHz NMR spectrometer (Varian, California, USA) at the oper-
ating frequencies of 300.13 and 125.67 MHz for proton and carbon
experiments, respectively. All the experiments were carried out at
25
C. Standard 1D 1H and 13C was carried out on all compounds
using the pulse sequences from the varian user library.
2.4. Cytotoxicity assay
2.4.1. Cell culture
Studies were carried out at the Drug Discovery unit at the fac-
ulty of Pharmacy, Mansoura University, Egypt. MCF-7 fibroblast cell
lines for MTT assay were cultured in DMEM supplemented with 10%
FBS (Fetal bovine serum) and 1% Pen-Strip at 37
C and 98% hu-
midity in a humidified incubator under an ambient pressure air
atmosphere containing 5% CO2, and allowed to achieve 80%
confluence. Confluent cells were then detached from culturing flask
wall using Trypsin EDTA solution (Trypsin EDTA; 200 mg/l versene
(EDTA), 170.000 U Trypsin/L) for 5 min and aliquots were collected
and sub-cultured. MCF-7 cells were seeded separately in 96-well
plate; [(BHK ¼ 1  103 cell/ml) within 100 mL/well DMEM sup-
plemented with 1% antibiotic and 10% FBS, and were incubated for
1 day to achieve cell attachment before adding the tested samples.
After incubation period, the medium in each well plate was dis-
carded and cells were incubated with different concentrations of
each tested compound as well as 5FU (5-Flouro-uracil) as positive
control. As a negative control group, the medium of cells (in some
wells) were changed to DMEM supplemented with 1% antibiotic
and 10% FBS and incubated 0.05% DMSO for 24 h (cells in this group
were not exposed to the test materials). Cells were incubated with
different extracts concentrations for 42 h prior to viability testing
with MTT assay.
2.4.2. Preparation of tested samples for cytotoxicity assays
Hexane fraction, essential oil, isolated compounds 1 (8-
methoxypsoralen), 2 (a-asarone) and 3 (3,4,5-trimethoxy-
Please cite this article in press as: N. Khalil, et al., Bioassay guided fractionation and cytotoxic activity of Daucus carota var. boissieri, Future
Journal of Pharmaceutical Sciences (2017), http://dx.doi.org/10.1016/j.fjps.2017.07.002
benzaldehyde) from red carrot fruits, as well as 5FU (5-Flouro-
Uracil) were prepared in four serial dilutions; 100, 50, 20 and 10 mg/
mL in DMSO 0.05% and complete growth media as vehicle before
being filtered through 0.22 mm micro-filter. Compounds (1 and 2)
showed solubility issue and produced slight turbidity upon media
addition for serial dilution as DMSO % got lower.
2.4.3. Cytotoxicity and MTT cell viability assay
The 3-(4,5-dimethyl-thiazoyl)-2,5-diphenyl-tetrazolium bro-
mide (MTT assay) was performed for the evaluation of MCF-7
fibroblast survival. This assay is based on the reduction of the sol-
uble yellow MTT tetrazolium salt to a blue insoluble formazan
product by mitochondrial succinic dehydrogenase.
For carrying out MTT assay, MTT solution was prepared as 5 mg/
mL MTT salts in complete medium (DMEM) just before use. MTT
solution was added to each well (10 mL in each well of 96-well
plates), then plates were isolated from light and incubated in a
humidified atmosphere of 5% CO2 in air at 37
C for additional 4 h.
When purple crystal formazan had formed around the cells, the
supernatant was carefully removed, and dimethylsulfoxide (DMSO)
was added to each well (100 mL/well in 96-well plates) and re-
incubated for 60 min to dissolve the dark blue formazan crystals.
Optical density values were measured using an ELISA BioTek
Lx800 microplate reader (BioTek, Bedfordshire, UK) at primary
wave length 570 nm (primary absorbance value) and a reference
wavelength of 630 nm (background absorbance value). The back-
ground absorbance was subtracted from the primary values and
cells viability was expressed with viability inhibition percentage (%)
which was calculated by using the following formula:
where, OD; Optical density.
2.5. Statistical analyses
All experiments were carried out in triplicates. Continuous
variable were presented as mean ± SD. The IC50 was determined as
the drug concentration which resulted in a 50% reduction in cell
viability or inhibition of the biological activity. IC50 values were
calculated using a four parameter logistic curve (SigmaPlot™ 11.0),
and all the data were statistically evaluated using student's t-test
(GraphPad Prism™ 5.01, GraphPad Software, Inc., CA, USA). The
criterion for the statistical significance was taken as P < 0.05.
3. Results
The hexane extract (HEX) was obtained as an oily greenish
brown residue and was dried over anhydrous sodium sulphate to
yield 128 g of oily extract (6.4% w/w) which was kept in sealed
container at 4
C until being used.
Approximately 10 g of the extract were put on a silca gel 60
column chromatography with isocratic elution using solvent sys-
tem hexane:ethyl acetate (70:30). Five main fractions were ob-
tained. Fraction I (2 g) was further subjected to a sub-column using
same stationary phase, and solvent system hexane:ethyl acetate
(80:20) to yeild 50 mg of compound (1). Fraction III (5 g) was
further purified on another sub-column with the same stationary
phase, and solvent system hexane:ethyl acetate (70:30) to yeild
70 mg of compound (2) and 40 mg of compound (3). The com-
pounds were then purified on another subcolumn until giving a
single spot with constant Rf on TLC.
 N. Khalil et al. / Future Journal of Pharmaceutical Sciences xxx (2017) 1e4 3

3.1. Structure elucidation of the isolated compounds
Compound (1) was obtained as creamy white fluffy needles
from the n-hexane fraction. It shows a single spot on TLC with Rf
0.34.
EI-MS of the compound exhibited a molecular ion peak at m/z
216.09 (Mþ). Further fragmentation in EI are given as follows: m/z
(% relative abundance) 201.08 (30), 173.06 (74),145.07 (44), 89.09
(100), 63.08 (71). IR (KBr): 1702, 1580, 1395, 1090 cm1. 1H NMR
(DMSO-d6, 300 MHz) spectral analyses gave signals at d ppm: 4.18
(s, 1H, CH3O), 6.43(d, J ¼ 9.6, 1H, 3-H), 7.1 (d, J ¼ 2.1, 1H, 40 -H), 7.68
(s, 1H, 5-H), 8.09 (d, J ¼ 2.1, 1H, 50 H), 8.24 (d, J ¼ 2.1, 1H, 4-H). 1H-1H
d ppm 191.84 (CHO), 153.27 (C-3, C-5), 142.79 (C-4), 131.6 (C-1),
106.68 (C-2, C-5), 60.19 (C-4 OCH3), 56.03 (C-3 OCH3, C-5 OCH3).
By comparing the above spectral data with the previously
published in literature [22], compound (3) was identified as 3,4,5-
trimethoxy-benzaldehyde. This compound was previously iso-
lated, once, from the roots of Beilschmiedia tsangii (Lauraceae) [23]
and also from other families like Fabaceae and Cactaceae, but iso-
lated for the first time from D. carota and from family Apiaceae. This
compound is close in structure to asaraldehyde (2,4,5-trimethoxy-
benzaldehyde); a compound previously isolated from D. carota var.
boissieri [20].

COSY (DMSO-d6) spectral analyses displayed the 1H-1H scalar 3.2. Cytotoxic studies
couplings, the most significant being H-3 4 H-4 and H-40 4 H-50 .
13
C NMR (DMSO-d6) spectral data showed carbon signals at d ppm The extract exhibited good cytotoxic activity shown through its
60.93(CH3O), 106.89 (C-40 ), 113.77 (C-50 ), 114.21 (C-3), 116.35 (C-4a), low IC50 (9.12 ± 0.58 mg/ml) against the standard 5-Flououracil
125.86 (C-6), 131.82 (C-8), 142.8 (C-8a), 145.21 (C-4), 147.79 (C-5), (8.46 ± 0.63 mg/ml), while the oil exhibited moderate activity
147.82 (C-7), 159.67 (C-2). (47.02 ± 1.73 mg/ml). Only compound (1) exhibited good cytotoxic
By comparing the above spectral data with the literature [15], activity (IC50; 9.38 ± 0.78 mg/ml), compound (2) had moderate
compound (1) was identified as 9-methoxyfuro[3,2-g]chromen-7- activity (46.12 ± 1.31 mg/ml), while compound (3) had no cytotoxic
one, also known as 8-Methoxypsoralen, Xanthotoxin, Ammoidin, activity (100.6 ± 3.11 mg/ml) (Table 1, Fig. 1).
Meladinine, Oxsoralen, Oxypsoralen.
This compound was previously isolated from D. carota [16,17].
Compound (2) was obtained as yellowish white crystals from 4. Discussion
the n-hexane fraction. It shows a single spot on TLC with Rf 0.77.
EI-MS of the compound exhibited a molecular ion peak at m/z Hexane extract of red carrot fruits exhibited good cytotoxic ac-
208.16 (Mþ). Further fragmentation in EI are given as follows: m/z tivity on MCF-7 fibroblast cell line shown through its low IC50
(% relative abundance) 193.14 (7), 165.09 (100), 132.11 (50), 43.09
(54). IR (KBr): 1606, 1509, 1460 cm1. 1H NMR (DMSO-d6, 300 MHz) Table 1
spectral analyses gave signals at d ppm: 6.99 (s, 1H, H-6), 6.54 (s, 1H, IC50 values for cytotoxicity of the isolated compounds of red carrot
H-3), 6.63 (dq, J ¼ 1.9, 15.9, 1H, H-7), 6.16 (dq, J ¼ 1.9, 15.9, 1H, H-8), fruits.
1.89 (dd, J ¼ 2, 6.7, 3H, H-9), 3.86 (s, 3H, 2-OCH3), 3.66 (s, 3H, 5- Compound no. IC50 value (mg/ml)
OCH3), 3.21 (s, 3H, 4-OCH3),. 13C NMR (DMSO-d6, 75 MHz) spec-
Hexane extract 9.12 ± 0.58
tral data showed carbon signals at d ppm 150.2 (C-4), 148.7 (C-2),
Compound (1) 9.38 ± 2.78
142.9 (C-1), 124.89 (C-7), 123.23 (C-8), 110.07 (C-5), 117.59 (C-6), Compound (2) 46.12 ± 1.31
98.42 (C-3), 56.2 (5-OCH3), 56.1 (2-OCH3), 55.7 (4-OCH3), 18.52 (C- Compound (3) 100.6 ± 3.11
9). 5-FU 9.46 ± 0.63
By comparing the above spectral data with the previously
published in literature [18], compound (2) was identified as (E)-
2,4,5-trimethoxy-1-(prop-1-en-1-yl)benzene, also known as a-
Asarone, trans-asarone, Etherophenol, Asaron, Azaron. The high J-
value for H-7 and H-8 (15.9) confirm the trans configuration of the
compound as previously reported by Singab in 1996 [19]. This
compound was previously isolated from D. carota [20,21]
Compound (3) was obtained as pale yellow crystals from the n-
hexane fraction. It shows a single spot on TLC with Rf 0.57.
EI MS of the compound exhibited a molecular ion peak at m/z
196.12 (Mþ). Further fragmentation in EI are given as follows: m/z
(% relative abundance) 109.11 (94), 77.1 (74), 44.06 (76), 43.09 (100).
IR (KBr): 1679, 1584, 1501 cm1. 1H NMR (DMSO-d6, 300 MHz)
spectral analyses gave signals at d ppm: 9.88 (s, 1H, CHO), 7.24 (s,
2H, H-2,6), 3.86 (s, 9H, -(OCH3)3).
13
C NMR (DMSO-d6)spectral data showed carbon signals at Fig. 1. IC50 values of tested samples relative to positive control (5-FU).

Please cite this article in press as: N. Khalil, et al., Bioassay guided fractionation and cytotoxic activity of Daucus carota var. boissieri, Future
Journal of Pharmaceutical Sciences (2017), http://dx.doi.org/10.1016/j.fjps.2017.07.002
4 N. Khalil et al. / Future Journal of Pharmaceutical Sciences xxx (2017) 1e4
(9.12 ± 0.58 mg/ml) against the standard 5-Flououracil
(8.46 ± 0.63 mg/ml). This cytotoxic effect could be attributed to
some specific component(s), as will be discussed later.
Fractionation of the hexane extract was needed to know
whether the isolated compounds would have the same cytotoxic
results or different ones. Three compounds were isolated using
column chromatography. A furanocoumarin identified as 8-
methoxypsoralen or xanthotoxin (Compound 1) and a phenyl-
propanoid identified as a-asarone (Compound 2). Those com-
pounds were previously isolated from the Daucus genus. The third
isolated compound (Compound 3) is also a phenylpropanoid
identified as 3,4,5-trimehoxybenzaldehyde, which shows a great
similarity in structure to a compound previously isolated from
Daucus and known as asaraldehyde (2,4,5-
trimethoxybenzaldehyde). This compound is reported here for
the first time to be isolated from this genus as well as from this
family.
The three isolated compounds, as well as the essential oil of the
red carrot fruits were also investigated for their cytotoxic activity
using MTT assay on MCF-7 cell lines. Only compound (1) exhibited
good cytotoxic activity (IC50; 9.38 ± 0.78 mg/ml), the essential oil
and compound (2) had moderate activity (47.02 ± 1.73 mg/ml and
46.12 ± 1.31 mg/ml respectively), while compound (3) had no
cytotoxic activity (100.6 ± 3.11 mg/ml). In a recent study, D. carota oil
proved to be selectively cytotoxic to human acute myeloid leukemia
cells [5]. In another study, significant cytotoxicity of isolated com-
pounds (mainly phenylpropanoids) from the essential oil of
D. carota against HL-60 cells was observed [24]. The effect of these
active chemicals altogether with other constituents of the hexane
extract should be considered in validating its use as an anticancer
drug.
These findings can validate the traditional use of carrot in the
management of carcinomas [25e27]. Xanthotoxin belongs to the
class of furanocoumarins which are considered as phototoxic and
photogenotoxic natural constituents occurring in a broad variety of
plants used in cosmetics, food and drugs but especially known for
their photosensitizing UV-induced action and used in many drugs
for the photodynamic destruction of tumor cells and other skin
disorders such as psoriasis and vitiligo [28]. Furanocoumarins are
powerful phytoalexins (scheel, 1986, Matern 1991), that's why
xanthotoxin concentration increased in infected carrots [17](Ceska,
1986). Xanthotoxin has been also found to give potent cytotxic
activity on MCF-7 cell lines [29]. The cytotoxicity of a-asarone and
its isomer b-asarone which are found together in certain plants
such as Acorus, Calamus and Asarum was investigated in HepG2-
cells and a-asarone was found to be more cytotoxic than b-asar-
one [30].
5. Conclusion
The hexaane extract of redcarrot fruits need to be further
explored for other promising cytotoxic compounds. The three iso-
lated compounds also need to be more investigated towards other
cell lines; also they are considered as a good substrate for future
SAR study and modifications to produce more potent cytotoxic
derivatives.
Role of funding source
This research did not receive any specific grant from funding
agencies in the public, commercial, or not-for-profit sectors.
Please cite this article in press as: N. Khalil, et al., Bioassay guided fractionation and cytotoxic activity of Daucus carota var. boissieri, Future
Journal of Pharmaceutical Sciences (2017), http://dx.doi.org/10.1016/j.fjps.2017.07.002
Conflict of interest
The authors declare no conflict of interest.
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