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Egypt. J. Chem. Vol. 62, No. 2 pp. 181 - 200 (2019)
*Corresponding author e-mail: amyoussef27@yahoo.com; Tel/ Fax, (202) 33322418 (202) 33370931
DOI: 10.21608/EJCHEM.2018.4960.1442
©2017 National Information and Documentation Center (NIDOC)
THIS STUDYCroton tiglium (C. tiglium) seeds
extracts by incorporation of silver nanoparticles (Ag-NPs) through raised up cytotoxicity
against growth of human colon cancer cells. C. tiglium seeds contain various phytoconstituents
protein was hydrolyzed in the dried aqueous seeds extract into free amino acids, the essential
amino acids (20.71%) & nonessential amino acids (79.29%). Furthermore, the mucilage in the
represented by total saturated fatty acids (13.68%), total monoenoic fatty acids (34.49%), total
(40.73%), unsaturated hydrocarbons (18.93%), fatty alcohols (10.08%), sterols (10.61%) and
O
from the petroleum ether extract. Additionally, incorporation of Ag-NPs into the extract
caused no toxicity on the experimental animals when administrated orally. It was found that
the median lethal dose (LD50) of the ethanolic, petroleum ether and aqueous seeds extract-Ag
nanocomposites was about 7.95, 5.2 and 65 ml/Kg, respectively.
Keywords: Croton tiglium L. Seeds, Silver nanoparticles, Polyphenols, Scavenging activity,
Anticancer activity.
17
Croton tiglium L. Seeds
Wael Mahmoud Aboulthana1, Ahmed M. Youssef2*, Amal M. El-Feky3
4, Mohamed M. Seif51
1Biochemistry Department, Genetic Engineering and Biotechnology Division,
National Research Centre, Dokki, Giza, Egypt (Afliation ID: 60014618).
2Packaging Materials Department, National Research Center, Dokki, Giza, Egypt
(Afliation ID: 60014618).
3Pharmacognosy Department, Pharmaceutical and Drug Industries Research
Division, National Research Centre, Dokki, Giza, Egypt (Afliation ID: 60014618).
4Microbial Biotechnology Department, Genetic Engineering and Biotechnology
Division, National Research Centre, Dokki, Giza, Egypt (Afliation ID: 60014618).
5Toxicology and Food contaminants, Food Industry and Nutrition Division, National
Research Center, Dokki, Giza, Egypt (Afliation ID: 60014618).
Croton tiglium L. belongs to the family
Euphorbiaceae that occur in tropical and
temperate regions all over the world and includes
about 280 genera and 8000 species which occur in
tropical and temperate regions all over the world
[1]. It is widely used in ethnomedicine for the
treatment of several cancer diseases [2]. Seeds,
C. tiglium are used in
traditional medicine for treatment of constipation,
dyspepsia, dysenteriae, gastrointestinal disorders,
visceral pain, headache, purgative, colds, fever,
worms, ascities and intracranial hemorrhage and
C. tiglium
(severe purgative action). This because the seeds
oil contains phorbol esters and crotonic acid along
with the fatty acids in addition to presence of
the active phytoconstituents. These constituents
are oil soluble and it is necessary to remove of
182
Egypt. J. Chem. 62, No. 2 (2019)
WAEL MAHMOUD ABOULTHANA et al.
reduce level of these constituents to reduce the
C. tiglium
process [6-8].
An attempt was made by High-performance
with other physiochemical parameters and
responsible for the severe purgative action of
C. tiglium seeds [7]. Moreover, the ethanolic
seed extract of C. tiglium exhibited strong
antidermatophytic activities. It was found that
crude extracts from these plant seeds were active
against Trichocomaceae (Aspergillusniger and
A. tamarii) and Mucoraceae (Mucormucedo and
Rhizopussolani) [9]. Thereafter, the ethanolic
seeds extract of C. tiglium exhibited atopical
formulation of the extracts into shampoo or soap
[10].
It was postulated that C. tiglium seeds extract
exhibited high antioxidant activity due to the
presence of some other phytochemicals such
as ascorbic acid, tocopherol and other pigments
[11]. 12-O-tetradecanoylphorbol-13-acetate is
major active constituent in the croton oil which
was isolated from seeds of this plant. It is used
for treatment of the solid tumors due to its ability
to inhibit the growth, stimulate apoptosis in
prostate, breast, colon and lung cancer diseases
[12]. In 2014, Yumnamcha et al. [13] emphasized
that aqueous C. tiglium seeds extract cause
dependent manner. They found that it is necessary
for the plant extract need to be evaluated before
it could be used for therapeutic purposes. The
recent study showed that C. tiglium seeds extracts
measurements but they have little effect on some
haematological indices [14].
Development of polymer-metal
nanocomposites containing metal nanoparticles
(MNPs) is considered to be one of the most
promising solutions to their inherent stability
problem. Incorporation of MNPs into polymeric
matrices showed valuable properties in many
practical applications [15-19]. Synthesis of silver
nanoparticles (Ag-NPs) by reduction of aqueous
silver nitrate into Ag-NPs during exposure to
plant extracts can be easily monitored by using
UV-visible spectrophotometer [20]. The plant
extracts with Ag-NPs exhibited good antioxidant
activity at lower concentrations [21]. Recently,
it was found that incorporation of Ag-NPs in
the plant extract increased the total phenolic
plant Ag-NPs showed a higher antioxidant and
antimicrobial activity compared to plant extract
alone or silver nitrate [22].
The present study aimed to evaluate the
different C. tiglium seeds extracts. Consequently,
after incorporation of the nanoparticles.
Materials and Methods
Preparation of the plant materials
The dried C. tiglium seeds were obtained from
Agricultural Research Center, Giza, Egypt and dried
in an incubator at 50 °C for 72 hrs. The dried seeds
were crushed into powder in an electric blender.
Pharmacopoeial constituents and vitamins (fat
and water soluble)
Moisture content was determined by heating
C. tiglium in the oven at
105C for 3hrs. Furthermore, the ashes contents
(total, water soluble and acid-insoluble ashes)
were assayed in the powdered seeds by method
soluble vitamins were analyzed according to
method suggested by Hasan et al. [24] using High
Performance Liquid Chromatographic system
(Shimadzu-UFLC Prominence), equipped with
an auto sampler (Model-SIL 20AC HT) and UV-
Visible detector (Model-SPD 20A).
Preliminary phytochemical screening tests
Wide range of the common phytoconstituents
was determined in the plant seeds according to
recommended and referenced methods. These
constituents were represented by carbohydrates
and /or glycosides [25], free and combined
nitrogenous compounds, sterols and /or
triterpenes [27], tannins [28], proteins [29] and
anthraquinones [30].
Preparation of different C. tiglium seeds extracts
for phytochemical studies
Preparation of alcoholic extract
The powdered seeds had been extracted
by cold maceration in successive portions of
80% ethyl alcohol at room temperature (cold
through a Whatman No. 1
concentrated to dryness in a rotary evaporator at
45°C under reduced pressure.
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EVALUATION OF ANTIOXIDANT EFFICIENCY OF CROTON TIGLIUM L. SEEDS ...
Preparation of petroleum ether extract
The dried seeds powder had been separately
extracted with petroleum ether (60-80°C). The
Whatman
No
in a rotary evaporator at 45°C under reduced
pressure.
Preparation of aqueous extract
The dried powdered seeds have been extracted
by distilled water. The extract was separately
Whatman No
then concentrated to dryness in a rotary evaporator
at 50°C under reduced pressure.
Chromatographic analysis of total phenolics
The different phenolic compounds
chromatograpahy (HPLC) (Shimadzu-UFLC
Prominence). This technique is consisting of two
units. One is responsible for separation of the
compounds according to the difference in polarity
and the other is responsible for detection of these
compounds. It is equipped with an auto sampler
(Model-SIL 20AC HT) and UV-Visible detector
(Model-SPD 20A) (Japan). The separation
process was carried out through analytical
column of an Eclipse XDB-C18 (150 X 4.6 µm;
5 µm) with a C18 guard column (Phenomenex,
Torrance, CA). The mobile phase is consisting
of solvent system of acetonitrile (solvent A)
and 2% acetic acid in water (v/v) (solvent B).
Before the chromatographic run, all samples were
(Gelman Laboratory, MI). Fifty micro litre (50 µl)
from each extract (alcoholic and water aqueous)
was injected automatically by the injector piece.
-1 for a total
run time of 70 min and the gradient program was
as follows: 100% B to 85% B in 30 min, 85% B to
50% B in 20 min, 50% B to 0% B in 5 min and 0%
simultaneously at 280 and 320 nm for the benzoic
acid and cinnamic acid derivatives, respectively.
times and UV spectra and compared with those of
the standards.
In the dried aqueous extract, total carbohydrate
sulphuric acid method [31]. Consequently, the
mucilagehas been separately isolated [32] then
34]. Furthermore, the Mucilage was hydrolyzed
Stephen [35]. The polysaccharides were analyzed in
the hydrolysate using Gas Liquid Chromatography
ionization detector at 270°C. The analysis was carried
14% cyanopropyl phenyl methyl, the carrier gas is
10.6 psi and velocity of 41 cm/sec. The injector
chamber temperature was -250°C. Quantitative
those of the authentic sugars.
In addition, the total protein content has
been estimated by determining the nitrogen
apparatus. The proteins were isolated, for isolation
method described by El-Gengaihi et al. [37]. The
dried crude polypeptides have been separately
dialyzed by membrane (dialysis bag) and the
non-dialyzable fraction was collected and dried.
Consequently, the polypeptides were hydrolyzed
into amino acids that have been separated by
HPLC then analyzed by amino acid analyzer
(model Eppendorf-Germany LC 3000) according
to method suggested by Widner and Eggum [38].
In the petroleum ether extract, the total
steroidal and terpenoidal contents were estimated
quantitatively by the spectrophotometric method
which is based on measuring intensity of the color
developed when sterols and triterpenes react with
Lieberman-Burchard reagent. Percentages of
steroidal and terpenoidal have been calculated
reference to a pre-established standard calibration
and concentrated according to method suggested
by Tsuda et al. [40].
Moreover, the total free fatty acid content
converted into the methyl ester form based on
method suggested by Finar [41]. Consequently,
matter were subjected to gas chromatograph
coupled with a mass spectrometer (model
Shimadzu GC/MS–QP5050A). The constituents
spectral fragmentation patterns with those of the
available database libraries Wiley (Wiley Int.)
USA and NIST (Nat. Inst. St. Technol., USA) and/
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Egypt. J. Chem. 62, No. 2 (2019)
WAEL MAHMOUD ABOULTHANA et al.
or published data [42]. Quantitative determination
The dried petroleum ether extract was
175 g activated silica (120 cm height x 2.5 cm
i.d.). Elution was successively carried out by
different ratios of petroleum ether (60-80°C) and
chloroform with increasing polarity. The fractions
were successively collected and concentrated
then screened by thin layer chromatography
(TLC) using benzene: ethyl acetate (8:2) as
solvent system. The TLC plates were examined
under UV-254 nm and visualized. The similar
fractions were combined then evaporated under
reduced pressure at a temperature not exceeding
40C. Thereafter, the isolated compounds were
subjected to physical, chemical, chromatographic
and spectral analyses (UV, MS, IR, 1H and 13C
NMR) as well as comparison with the available
reference standards and available published data.
Preparation of C. tiglium seeds extracts silver
nanocomposites
The Ag-NPs were prepared through reducing
silver nitrate (AgNO3) with ethylene glycol (EG)
in the existence of polyol. In a classic method, 10
Then, 5 mL solution of AgNO3 in EG and 10 mL
solution of 0.15 M PVP in EG containing 0.03 mM
MnCl2
over a period of 10 min, the reaction mixture was
for 60 min. The reaction mixture was then cooled
to room temperature. The product was centrifuged
at 3000 rpm for 5 min, and then washed with
acetone and ethanol for three times. The Ag-NPs
the Ag-NPs were added to the C. tiglium seeds
extracts by different concentrations to form C.
tiglium seeds extracts silver nanocomposites.
In vitro antioxidant and cytotoxic activities of the
different extracts
Total polyphenolic compounds
The total polyphenolic compounds were
estimated in the different C. tiglium seeds
extracts before and after incorporation of silver
nanoparticles according to Singleton and Rossi
[43] by using folin ciocalteu reagent purchased
from Sigma Chemicals Co. Concentration of the
total polyphenols was calculated as a gallic acid
equivalent from the calibration curve of gallic
acid standard solutions obtained from Sigma
Chemicals Co. covering the concentration range
between 0.2 and 1.0 mg / ml.
Total antioxidant capacity
Total antioxidant capacity of extract was
evaluated through the assay of the green phosphate/
Mo5+ complex according to the method described
by Prieto et al. [44]. Antioxidant capacity was
expressed as mg gallic acid equivalent per gram
dry weight.
Total reducing power
The total reducing power was determined
according to method suggested by Oyaizu
[45]. The absorbance was measured at 700nm
Ascorbic acid at various concentrations was used
as standard. A high absorbance of the reaction
mixture at 700nm indicates a higher reducing
power.
Free radical scavenging activity
DPPH radical-scavenging activity
Percentage of the antioxidant activity was
evaluated by method described by Brand-
Williams et al. [46] using DPPH (2,2-diphenyl-1-
picryl-hydrazyl-hydrate) for initiation of the free
radicals and absorbance of the resulting solution
was measured spectrophotometrically at 517 nm.
ABTS radical scavenging assay
For 2, 2’-azinobis-(3- ethylbenzothiazoline-
6-sulfonic acid) (ABTS) assay, the procedure
followed the method suggested by Arnao et
al
were allowed to react with ABTS solution and
using a spectrophotometer. The ABTS scavenging
capacity of the extract was compared with that of
ascorbic acid.
Anticancer activity
Cytotoxic activity test (In vitro bioassay
on human tumor cell lines) was conducted
and determined. It was performed on human
colon carcinoma cell line according to protocol
Characterization of the prepared Ag-NPs
X-ray diffraction (XRD)
determined using a Philips X-ray diffractometer
(PW 1930 generator, PW 1820 goniometer)
0.02 and step time of 1s.
Transmission Electron Microscope (TEM)
The morphological and particles size of
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Egypt. J. Chem. 62, No. 2 (2019)
EVALUATION OF ANTIOXIDANT EFFICIENCY OF CROTON TIGLIUM L. SEEDS ...
prepared samples were demonstrated by using
TEM model JEM-1230, Japan, operated at 120
3 and
a resolution until 0.2 nm. A drop of an aqueous
dispersion of the prepared samples was placed on
a carbon-coated copper grid and allowed to dry in
air before characterization.
UV-spectroscopy of the prepared Ag-NPs
UV-spectroscopy was carried by Shimadzu
UV-Vis recording spectrophotometer UV-240.
DLS measurements
Particle size distribution was measured using
Particle Sizing Systems, Inc. Santa Barbara,
Calif., USA.
Ethical Statement
The experimental design and animal handling
were performed according to the experimental
protocol which was approved by Institutional
Animal Ethics Committee of National Research
in accordance with guidelines as per “Guide for
the care and use of laboratory animal” and with
permission from Committee for the Purpose
of Control and Supervision of Experiments on
Animals.
Median lethal dose of different extracts (LD50)
The different C. tiglium seeds extracts
(after incorporation of Ag-NPs) were evaluated
separately after calculating the LD50. Two hundred
and forty adult albino mice (weight 20-25 g) were
used to study acute toxicity. Animals were divided
into 10 groups (8 mice in each group) for each
extract. The groups were treated orally by stomach
tube with rising doses of 0.2, 0.4, 0.6, 0.8, 1.0,
1.2, 1.4, 1.6, 1.8 and 2.0 ml/mice of extract-Ag
nanocomposites. Mortality was recorded after 24
hrs of extract treatment. The LD50 was calculated
for each extract-Ag nanocomposites using
equation suggested by Paget and Barnes [49].
Result and
Evaluation of phytochemical and physico-
chemical properties of C. tiglium seeds
The phytochemical and physico-chemical
evaluation of C. tiglium seeds provide useful
information to verify the pharmacognostical
identity of seeds of this plant and to carry
phytochemical and physicochemical
investigations of these seeds [50].
Table 1 revealed data of the physiochemical
parameters (moisture, total ash content, water-
soluble ash and acid insoluble ash) which
determined in the dried C. tiglium seeds
powders following procedure of the Egyptian
pharmacopoeia. These constants could be
purity of these plant seeds. Elevation of the ash
attributable to loss of oil soluble constituents in
the plant seeds [51].
with complex mixtures of biologically active
compounds, some of the compounds in such
a mixture can be genotoxic or antigenotoxic
[13]. For this reason, it is necessary to undergo
phytochemical screening to predict the potential
health hazards occurred as a result of using the
plant for medicinal purposes. Phytochemical
constituents are non-nutrient, bioactive, secondary
metabolites, naturally occurring plant compounds
present in C. tiglium seeds and are considerably
diverse [52].
Phytochemical screening of C. tiglium seeds
These constituents are commonly used for
medicinal purposes as analgesic, antimalarial,
bactericidal and antiseptic [53]. In the present study,
the air-dried C. tiglium seeds powder subjected to
the phytochemical screening tests for the detection
of various bioactive compounds using chemical
methods. As illustrated in Table 2, the air-dried
seeds powders contain various phytoconstituents
compounds) and proteins. This was in accordance
with the study documented by Lopes et al. [54]. It
was speculated that certain saponin mixture and
effects of C. tiglium seeds.
It was reported that certain group of bioactive
are responsible for the DNA damage [55]. Further
studies with more advanced practical techniques
of C. tiglium seeds. Moreover, Salatino et al. [52]
reported that terpenoids are the predominant
secondary metabolite constituents in seeds of
this plant. Triterpenoids, either pentacyclic or
steroidal, have frequently been reported for
seeds of Croton species. From medicinal point of
view, all the seeds extracts revealed the presence
glycosides, tannins, glycoside and saponins which
are considered very important components [56].
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Egypt. J. Chem. 62, No. 2 (2019)
WAEL MAHMOUD ABOULTHANA et al.
C. tiglium seeds.
Moisture Total Ash Water soluble ash
7.5 15 8.6 5.7
TABLE C. tiglium seeds.
Constituent Result
Carbohydrates (Glycosides) ++
Flavonoids +
Saponins -
Tannins -
Sterols (Triterpenes) ++
+
Proteins +
Anthraquinones -
(-): Absent, (+): Present, (++): Appreciably present.
Presence of carbohydrate in the seeds extract could
be a good source of energy [57]. Also, presence
C. tiglium seeds with
high quantity may be responsible for antiemetic
activity. Saponins are responsible for antibiotic
inhibit growth of cancer cells [58, 59]. Flavonoids
aggregation, allergies and microbial infection
[60].
Study the physical and chemical characters of the
C. tiglium seeds
As presented in Table 3, it was found that the
highest yield was obtained in petroleum ether
extract which appeared oily yellowish brown
extract before incorporation of Ag-NPs, this
because the C. tiglium seeds could be a good
Also, petroleum ether extract contains the highest
by the study carried out by Lan et al. [61] who
reported that the oil obtained by extracting of
the C. tiglium seeds with petroleum ether was
ethyl ether. They postulated that the linoleic acid,
oleic acid and eicosenoic acid were the main
samples in addition to the aromatic compounds
(phorbol esters). Ganer et al. [50] emphasized that
toxicity of C. tiglium seeds might refer to presence
of oil soluble phorbol esters and crotonic acid
in addition to the other constituents. During the
removal of these constituents. Thereafter, seeds
of this plant became suitable for the therapeutic
purposes.
and Vit. B1 are the most abundant fat and water
soluble vitamins in C. tiglium seeds. Moreover,
concentrations of Vit D3 and Vit B3 were too
low to be detected. This was in accordance with
Bello et al. [57] who mentioned during their study
that the seeds could be good source of fat soluble
vitamins.
HPLC Analysis of the free amino acids in the
dried aqueous C. tiglium seeds extract
It was found that the dried aqueous C. tiglium
seeds extract contains protein content representing
about 25% (wt/wt) of the dried extract [38]. After
of the protein was about 23.5% (wt/wt) dried
aqueous extract [37]. As presented in Table 5,
there were eight essential and nine non-essential
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EVALUATION OF ANTIOXIDANT EFFICIENCY OF CROTON TIGLIUM L. SEEDS ...
TABLE C. tiglium
C. tiglium
Petroleum ether Aqueous
Percentage (%) 31 45 23
Physical characters:
Color Brown Yellowish brown Brown
Condition Semi-solid Oily Semi-solid
Constituents:
Carbohydrates + - +
Flavonoids + - +
Saponins - - -
Tannins - - -
Sterols + ++ -
+ - +
Proteins + - +
Anthraquinones - - -
(-): Absent, (+): Present, (++): Appreciably present.
TABLEC. tiglium seeds.
Fat soluble vitamins
Vit A 0.646
Vit D3 ND
9.495
Water soluble vitamins
B3 ND
B1 298.149
B6 0.288
B9 0.695
B2 1.328
B12 0.479
ND: Not detected concentration.
C.
tiglium seeds extract representing about 20.71%
(wt/wt) and 79.29% (wt/wt) of the total amino
acids, respectively. Moreover, it was found that
isoleucine, methionine and phenyl alanine belong
to the major essential amino acids representing
about 4.74, 3.84 and 3.54% (wt/wt) of the total
amino acids content, respectively. However,
proline and aspartic acid belong to the major non-
essential amino acid and represent about 14.41
and 13.32% (wt/wt) of the total amino acids
content, respectively.
GLC analysis of mucilage after hydrolysis in the
dried aqueous C. tiglium seeds extract
The carbohydrate content was estimated
quantitatively in the dried aqueous C. tiglium seeds
extract [25] and it was found that it represented
about 16% (wt/wt) of the dried aqueous extract
[31. Based on the analysis suggested by Evan and
Matz [33, 34], it was noticed that no gelatinous
precipitate appeared upon the reaction with
potassium hydroxide. In addition, the red stain
obtained as a result of the reaction with ruthenium
red indicates the mucilaginous nature with
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Egypt. J. Chem. 62, No. 2 (2019)
WAEL MAHMOUD ABOULTHANA et al.
TABLEC. tiglium
Threonine 1.59 Aspartic acid 13.32
Valine 2.42 Serine 11.30
Methionine 3.84 Glutamic acid 9.53
Isoleucine 4.74 Glycine 7.87
Leucine 1.63 Proline 14.41
Phenyl alanine 3.54 Alanine 4.02
Lysine 2.03 Tyrosine 10.85
Tryptophan 0.92
Histidine 4.74
Arginine 3.25
Total essential amino acids 20.71 Total non essential amino
acids 79.29
eliminating pectin. Furthermore, it was found the
isolated mucilage expressed by 14.5% (wt/wt) of
the dried aqueous extract. As compiled in Table
6, composition of the mucilage was determined
qualitatively and quantitatively in the hydrolyzate
by GLC.
GC/MS analysis of the saponiable matter in the
petroleum ether C. tiglium seeds extract
spectrophotometrically in the petroleum ether
C. tiglium seeds extract [39]. It was found that
the total steroidal and terpenoidal contents were
expressed by 12% and 24 % of the dried extract
compiled in Table 7 showed the methylated ester
ether C. tiglium seeds extract by GC/MS. It
was revealed that eighteen compounds were
constituted about 13.68%, while the unsaturated
fatty acids constituted 80.25%. Furthermore,
it was found that 11,14-Octadecadienoic acid
(26.57%) was the major unsaturated fatty acid,
while Hexadecanoic acid (6.64%) was the major
saturated fatty acid.
TABLEC. tiglium
Arabinose 8.31 23.38
Xylose 8.62 15.21
Ribose 8.97 11.84
Rhamnose 9.31 9.18
Galactose 13.29 13.15
Glucose 14.38 8.45
81.21
R.T: Retention time.
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EVALUATION OF ANTIOXIDANT EFFICIENCY OF CROTON TIGLIUM L. SEEDS ...
TABLE C. tiglium
Rt Mol.
Base
14.79 5.04 9-Tetradecenoic acid 240 55 69, 74, 83, 110, 125, 137, 166, 208
15.43 1.05 3-Methyl-tetradecanoic acid 256 74 83, 101, 111, 129, 143, 156, 213, 225, 241
15.90 6.64 Hexadecanoic acid 270 74 87, 97, 129, 143, 199, 227, 239
17.64 1.04 3-Methyl-hexadecanoic acid 284 74 87, 101, 115, 157, 171, 213, 227, 241,
253, 270
19.73 8.10 9,11-Octadecadienoic acid 294 67 81, 95, 109, 123, 150, 164, 263
19.98 26.57 11,14-Octadecadienoic acid 294 67 81, 95, 109, 150, 164, 178, 263
20.09 13.84 9-Octadecenoic acid 296 55 69, 74, 83, 98, 111, 123, 137, 180, 222,
264
20.46 2.76 Octadecanoic acid 298 74 87, 97, 129, 143, 185, 199, 213, 255
21.84 1.06 Nonadecanoic acid 312 74 87, 97, 111, 129, 143, 157, 185, 199, 213,
269
22.00 2.04 5-Eicosenoic acid 324 55 67, 74, 96, 110, 123, 152, 180, 208, 250,
275, 292
22.77 3.06 11-Docosenoic acid 352 55 69, 74, 83, 97, 123, 152, 180, 208, 236,
263, 278, 292
23.24 0.09 Docosanoic acid 354 74 87, 97, 129, 143, 157, 199, 213, 255, 311,
323
23.95 4.26 15-Tetracosenoic acid 380 55 69, 74, 83, 97, 111, 123, 152, 194, 207,
222, 250, 264, 277, 291, 306, 348
24.31 5.09 16-Pentacosenoic acid 394 55 69, 74, 83, 97, 111, 125, 152, 194, 222,
236, 264, 278, 291, 320, 344, 362
24.68 1.04 24-Methyl-pentacosanoic acid 410 74 87, 97, 111, 129, 143, 157, 199, 213, 255,
269, 311, 325, 353, 367
26.13 6.05 5,9-Heptacosadienoic acid 420 81 95, 109, 141, 150, 164, 181, 207, 222,
278, 346, 371, 388
26.50 5.04 5,9-Octacosadienoic acid 434 81 95, 109, 141, 150, 163, 181, 207, 236,
264, 292, 319, 360, 385, 402
28.86 1.16 19-Octacosenoic acid 436 55 69, 74, 83, 97, 111, 125, 152, 194, 222,
236, 264, 292, 320, 362, 387, 406
Total saturated fatty acids: 13.68%
Total monoenoic fatty acids: 34.49%
Total dienoic fatty acids: 45.76%
Rt: Retention time
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Egypt. J. Chem. 62, No. 2 (2019)
WAEL MAHMOUD ABOULTHANA et al.
GC/MS analysis of the unsaponiable matter in
the petroleum ether C. tiglium seeds extract
Table 8 displayed components of the
C.
tiglium seeds extract by GC/MS. It was found that
matter. In addition, n-Docosane (14.42%) and
7-Phenyltridecane (9.10%) were expressed as
During the present study, four compounds were
isolated from the petroleum ether C. tiglium seeds
TLC using different developing solvent systems.
Consequently, these isolated compounds were
characterized with recording their Rf values in
benzene: ethyl acetate (8:2) accurately. All of
the isolated compounds gave positive result with
Lieberman-Burchard test indicating the presence
of triterpenes and sterols [62]. Structures of these
TABLE C. tiglium
Rt Mol.
Base
9.71 0.50 2,3-Dimethylnonane 156 57 43, 71, 83, 112, 141
11.96 0.61 n-Dodecane 170 57 43, 71, 85, 99, 113
18.78 0.97 2,6,11-Trimethyldodecane 212 57 43, 71, 85, 99, 127, 141,169
19.43 0.32 5-phenyl Decane 218 91 105, 119, 147, 161
21.23 0.70 2-phenyl Decane 218 105 119, 131, 181
21.87 1.42 6-phenyl undecane 232 91 105, 119, 161, 189
21.98 4.00 6-phenyl dodecane 246 91 105, 119, 147, 189
22.26 3.20 1-Hexadecanol 242 55 69, 83, 97, 111, 125, 196, 224
22.81 2.80 2-Phenyldodecane 246 105 119, 161, 207, 218
24.32 9.10 7-Phenyltridecane 260 91 105, 119, 133, 175, 189
24.74 5.89 2-Phenyltridecane 260 105 119, 147, 161, 190, 232, 245
25.28 4.75 1-Nonadecene 266 43 55, 83, 97, 111, 125, 139, 168
26.24 7.67 2-Methyl7-nonadecene 280 43 57, 69, 83, 97, 111, 125, 252
26.62 14.42 n-Docosane 310 43 57, 71, 85, 99, 113, 127, 239
26.77 6.88 1-Docosanol 326 43 55, 69, 83, 97, 111, 125
27.05 3.47 398 398 199, 213, 255, 271, 300, 337, 355,
365
27.60 2.89 400 43 213, 255, 273, 327, 357
28.51 4.25 414 414 199, 213, 231, 241, 255, 273, 303,
329, 339, 367, 381, 396
39.48 6.51 17-Pentatriacontene 490 43 57, 83, 97, 111, 125, 139, 462
Saturated Hydrocarbons: 40.73%
Unsaturated Hydrocarbons: 18.93%
Fatty alcohols: 10.08%
Sterols: 10.61%
Rt: Retention time
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EVALUATION OF ANTIOXIDANT EFFICIENCY OF CROTON TIGLIUM L. SEEDS ...
compounds have been elucidated by comparing their
pectroscopical results with those published data.
Study of structure of the isolated compounds
from petroleum ether : chloroform (75:25) as white
crystal, Rf 0.69, melting point 133-134 °C which
in agreement with that reported in literature[63],
UVmax: 203, m/z (relative intensity): 414
(54%) for the molecular formula C29H50O, and
m/z 43, in addition to other
to (M-45) due to loss of HO+3, also, 271
(22%) as a result of the formation of carbonation
loss of C10H23 that corresponds to the M-143,
beside to 396 (28%) due to the loss of a water
molecule and 133 (18%), 105 (23%), 107 (26%),
95 (31%), 81 (34%), 69 (32%), 55 (41%).
The absorption spectrum (KBr, cm)
Stretch), 1388 (Aromatic C-H Bending), 1036 (C-
OH Stretch) and 885 (O-H Bending).
1 (δ values, CDCl3): 5.29 (1H,H-6)
owing to the double bond between C-5 and C-6,
the presence of two singles at 0.66 (3H,s, Me-18)
and1.12(3H,s,Me -19) refer to the angular methyl
groups C-18 and C-19. Also the appearance of
three doublets at δ 1.04(3H,d,Me-21),0.83(3H,d,
JJ
and one triplet at 0.86(3H,t, J
C-29 methyl groups, respectively.
13: δ 11.89 (C-18), 11.98(C-29),
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WAEL MAHMOUD ABOULTHANA et al.
18.69(C-21), 18.82(C-27), 19.34(C-19), 19.56(C-
26), 21.42(C-11), 23.18(C-28), 24.35(C-15),
26.31(C-23), 28.36(C-16), 29.27(C-25), 31.64(C-
2), 31.94(C-7), 32.17 (C-8), 32.48(C-22),
34.15(C-20), 36.35(C-10), 36.75(C-1), 37.45(C-
4), 39.79(C-12), 42.36(C-13), 45.64(C-24),
50.13(C-9), 56.21(C-17), 56.72(C-14), 77.31(C-
3), 121.46 (C-6), 140.72(C-5).
Compound 2 (-amyrin) (Fig. 1b): isolated
from petroleum ether : chloroform (50:50) as
white needle crystals, Rf 0.66, melting point
185-186 °C as stated by Siratet al. [64]. UV data
displayed characteristic absorption bands at 275,
260, 243 nm, m/z (relative intensity)
shows molecular ion at m/z 426 (30%) which is
consistent with the molecular formula C30H50O
to other characteristic fragments at 43 (57%), 55
(43%), 57 (31%), 71 (23%), 95 (63%), 127 (24%),
175 (13%), 189 (11%), 203 (28%), 257 (9%), 293
(13%), 409 (7%), 411 (18%).
The spectrum (KBr, cm) showed absorption
2724 (C–H in conjugation), 1652 (ethylenic double
C-H Bending), 1124, 1021 (C-OH Stretch), 974, 886
(O-H bending).
1 (δ values, CDCl3): 0.84 (s, 3H, –
CH3), 0.89 (d, 3H, J3), 1.06 (d, 3H,
J 3), indicating the triterpenoidal
nature, 1.27–1.30 (d, 6H, J
–CH3
(s, 3H, –CH3 at H-29) 1.49 (m, 2H, H-16), 1.53
(m, 2H, H-21), 1.59 (d, 1H, J
1.67 (s, 7 H, –CH3 × 2 and H-5), 1.78 (m, 1H,
H-19), 1.80 (m, 2H, H-22), 1.89 (m, 2H, H-15),
2.02 (d, 2H, J
2.20-2.27 (d, 2H, J
3.49 (s, 2H, H-7), 3.59 (m, 2H, H-2), 5.09 (s, 2H,
H-11), 5.16 (s, 1H, H-12).
13
17.6 (C-29), 18.5 (C-6), 21.6 (C-30), 23.2 (C-27),
23.5 (C-11), 26.5 (C-16), 27.1 (C-15), 28.2 (C-23,
C-28), 28.7 (C-2), 31.3 (C-21), 32.4 (C-7), 33.9
(C-17), 36.7 (C-10), 38.8 (C-1, C-4), 39.6 (C-19,
C-20), 40.6 (C-8), 41.4 (C-22), 42.1 (C-14),47.8
(C-9), 55.2 (C-5), 59.2 (C-18), 79.7 (C-3), 124.6
(C-12), 139.4 (C-13).
Compound 3 (Oleanolic acid) (Fig. 1c):
isolated from petroleum ether : chloroform
(50:50) as white amorphous solid, Rf 0.62, melting
point at 173 °C as stated in Galgon et al. [66].UV
max at 205 nm, m/z (relative intensity):
457 (43%) for the molecular formula C30H48O3,
m/z 439 characteristic
double bond, 411 (59%), 247 (61%), 203 (23%),
191 (14%), 177(24%).
The spectrum (KBr, cm) showed
(Aromatic C-H Bending), 1184, 1161, 1030 (C-
OH Stretch), 990, 865 (O-H bending).
1(δ values, CDCl3): 0.77, 0.88, 0.89,
0.92, 0.96, 1.07 and 1.32 (7s, 21H, all –CH3),
1.38 (m, 2H, H-21), 1.40 (m, 2H, H-16), 1.54
(m, 5H, H-18, H-19 and H-15), 2.11 (m, 3H, H-1
and H-9), 3.20 (t, 1H, J
2H, H-7), 4.55 (s, 2H, H-11), 4.62 (s, 1H, H-12).
1
proton resonating at δ 4.62 (1H) (typical of oleane
13
(C-26), 18.11 (C-6), 22.78 (C-11), 23.13 (C-30),
23.48 (C-16), 25.68 (C-27), 26.94 (C-2), 27.49
(C15), 28.01 (C-23), 30.47 (C-20), 32.24 (C-22),
32.54 (C-7), 32.97 (C-29), 33.69 (C-21), 37.01 (C-
10), 37.89 (C-1), 38.52 (C-4), 39.24 (C-8), 41.02
(C-18), 42.13 (C-14), 45.76 (C-17), 46.23 (C-19),
47.85 (C-9), 54.79 (C-5), 79.34 (C-3), 121.89 (C-
12), 144.51 (C-13), 182.03 (C-28). The presence
13C-NMR
data with the signals in the region δ 15.11 ppm,
at δ 121.89 and δ144.51 attributed respectively
to seven methyl groups, to C-12, C-13 and 12–
Compound 4 (3-O--D-glucopyranosyl--
sitosterol (daucosterol)) (Fig. 1d): isolated from
petroleum ether : chloroform (25:75) as white
crystals, Rf 0.58, melting point 281-282°C
which in agreement with that stated byFaiziet
al. [67].UV max: 205 and 197 nm. m/z
(relative intensity): 576 (36%) for the molecular
formula C35H60O6 m/z
corresponding to (M+-glycosidic unit), 396
(47%) indicated the loss of a water molecule
from β-sitosterol nucleus, beside 381 (34%), 329
(26%), 303(18%), and 275 (24%).
The absorption spectrum (KBr, cm)
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EVALUATION OF ANTIOXIDANT EFFICIENCY OF CROTON TIGLIUM L. SEEDS ...
Stretch), 1385 (Aromatic C-H Bending), 1055 (C-
OH Stretch) and 892 (O-H bending).
1 (δ values, CDCl3): 5.34 (1H, br, H-6)
J
Hz, H-1’), 3.54 (1H, m, H-3), and 3.41(1H, d,
H-2’).
13: δ ppm11.4 (C-18), 11.9 (C-29),
18.2 (C-21), 18.7 (C-27), 19.1 (C-19), 19.4 (C-
26), 20.6 (C-11), 21.9 (C-28), 25.0 (C-15), 26.2
(C-23), 28.5 (C-16), 28.9 (C-2), 29.6 (C-25), 30.8
(C-7/8), 33.6 (C-22), 35.4 (C-20), 36.0 (C-10),
36.7 (C-1), 37.8 (C-4), 39.6 (C-12), 42.1 (C-13),
44.9 (C-24), 49.3 (C-9), 55.2 (C-17), 56.4 (C-14),
61.3 (C-6’), 71.2 (C-4’), 73.7 (C-2’), 76.9 (C-3),
77.4 (C-3’/5’), 100.6 (C-1’), 121.4 (C-6), 140.3
(C-5). The acid hydrolysis of the compound led
to the presence of glucose in aqueous layer and
with authentic reference samples in addition to
O-glucopyranoside.
Among the all noble metal nanoparticles,
the Ag-NPs gained boundless interests due to
their characteristic properties in addition to their
exhibits vital functions as an antiseptic and
displays a broad biocidal effect against various
microorganisms through disruption of their
unicellular membrane thus disturbing their
enzymatic activities [69, 70]. Many reports
have been published concerning synthesis
of Ag-NPs using plant extracts revealed that
the nanoparticulated extracts were economic,
communities, protecting human health and
environment, leading to less waste and more
safe products [71-73]. For this reason, during
the current study, they can be incorporated into
C. tiglium seeds extract to raise its antioxidant
Assessment of the structure and morphology of
fabricated nanomaterials
The XRD as well as TEM consider the most
properties of the fabricated nanomaterials. The
Ag-NPs were prepared and studied using the
XRD diffraction pattern. As revealed in Fig. 2a, it
was noticed that XRD result of the prepared Ag-
Ago found at 37.8°, 44.5° and 67.6° matching with
the crystallographic planes (1 1 1), (0 0 2) and (0 2
2) of Ag-NPs, respectively and generates a typical
of crystalline metallic Ag phase.
Also, XRD displayed separate diffraction
(002) of the cubic face-centered silver. These
using for stabilizing the prepared Ag-NPs. Strong
scattering centers in the crystalline phase and
might be because of capping agents. An increase
in the incubation time with Ag-NO3 solution
along with plant extract the synthesis of Ag-NPs
increased. Presence of the plant extract reduced
formation of AgNO3 into Ag ions. Moreover, the
secondary metabolites present in the extract act
as a reducing and a capping agent for Ag-NPs
synthesis [74-78].
In order to assess the shape, size and
morphology of nanoparticles, TEM was used. It
was revealed that the Ag-NPs were well dispersed
and were predominantly spherical in shape, while
some of the NPs were irregular in shape. Based on
the obtained morphological data using TEM (Fig.
2b), it was demonstrated that the Ag-NPs was
formed as a result of the chemical reduction which
was carried out in presence of AgNO3 solution.
Presence of Ag nanoparticles maintained the
homogeneity and uniformity of the distribution
of Ag-NPs in the particles size range (5-10 nm)
as revealed from TEM image. It was revealed
that the Ag-NPs were well dispersed and were
predominantly spherical in shape, while some
of the NPs were irregular in shape. This was in
[79]. Furthermore,
evidence for the formation of Ag-NPs prepared
within the chemical reduction method was
demonstrated using UV-visible spectroscopy. This
technique is a precise suitable and reliable method
for the main characterization of manufactured
nanoparticles which is correspondingly used to
display the Fabrication as well as stability of Ag-
NPs.
The Ag-NPs have exceptional optical
properties that generate strongly interrelate
with exact wavelengths of light. Also, UV-Vis
spectroscopy is easy, fast, simple and discerning
for various types of NPs, requests simply a short
period time for measurement. As illustrated in
at 450 nm that reveals the formation of Ag-NPs.
Consequently, DLS is mostly used to determine
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WAEL MAHMOUD ABOULTHANA et al.
particle size as well as size distributions in
aqueous solutions. The size attained from DLS is
frequently larger than that particle size obtained
from TEM, which might be because of the effect
of Brownian motion. DLS is a nondestructive
technique used to acquire the average diameter of
the prepared dispersed nanoparticles in aqueous
solutions. As presented in Fig. 2d, it was found
that the particle size distribution of the fabricated
Ag-NPs has main diameter around 82 nm.
The plants were effectively involved in
synthesis and controlled formation of Ag-
NPs. The nanoparticles size differs from TEM
to DLS and this may be the reason of particles
agglomeration. Furthermore, the nanoparticles
exhibited good stability and this might be due to
presence of secondary metabolites as a capping or
reducing agent [80].
Total antioxidant capacity and free radical
scavenging activity in different C. tiglium seeds
extracts before and after incorporating Ag-NPs.
Polyphenols are the most common biologically
active molecules. They are structurally
characterized by presence of one or more phenol
units. They are considered as one of the most
important classes of secondary plant metabolites
that play an important role in prevention of chronic
diseases owing to their antioxidants potentials [81
& 82]. As illustrated in Table 9, it was found that
the aqueous C. tiglium seeds extract contains the
highest concentration of polyphenolic compounds
(699.21± 6.93 mg gallic acid/100 gm) as compared
to the other extracts. This was in agreement with
the study suggested by Salatino et al. [52] who
reported that these phenolic compounds were
proanthocyanidins predominate. It was shown
that the nanoparticulated extracts exhibited
elevated concentrations on total polyphenolic
compounds in ethanolic, petroleum ether and
aqueous C. tiglium seeds extracts (873.54 ± 9.31,
485.91 ± 5.18 and 962.05 ± 10.25 mg gallic
acid/100 gm, respectively) than the crude extracts
(617.87 ± 3.85, 388.24 ± 6.47 and 699.21 ± 6.93
mg gallic acid/100 gm, respectively). This was in
accordance with Abdelhady and Badr [83] who
reported that incorporation of Ag-NPs increased
concentration of the polyphenolic compounds
with respect to the native extract (without Ag-
NPs).
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Egypt. J. Chem. 62, No. 2 (2019)
EVALUATION OF ANTIOXIDANT EFFICIENCY OF CROTON TIGLIUM L. SEEDS ...
In addition, it was noticed that the aqueous C.
tiglium seeds extract proved promising antioxidant
capacity, total reducing power and free radical
scavenging activity by 0.856 ± 0.019 mg gallic
acid / gm, 8.81 ± 1.10 and 73 %, respectively. The
antioxidant properties of the seeds extract might be
attributed to presence of the main components of
and guaiol) [84]. In addition, the aromatic acids
(vanillic and 4-hydroxy-benzoic acid along
antioxidant activity [85]. Furthermore, it was
noticed that incorporation of Ag-NPs enhanced
the antioxidant properties through increasing the
total antioxidant capacity, total reducing power
and free radical scavenging activity in comparison
with the native extracts. This was in accordance
with Abdelhady and Badr [83] who suggested
that nanoparticulated extracts increased levels of
the active phytoconstituents which exhibit more
free radical scavenging activity than the crude
extracts.
The in vitro anticancer activity showed that
the seeds powder and ethanol extract exhibited
equal anticancer activity against human colon
cancer cells with IC50
accordance with Mohd Ali et al. [86] who reported
that the ethanolic Croton extracts exhibited high
antioxidant activity by means of DPPH radical
scavenging activity, reducing power and total
antioxidant capacity. Furthermore, this might
be due to the highest total phenolic and total
use of C. tiglium seeds extract as a natural source
of antioxidant. Petroleum ether extract showed no
anticancer activity. This might refer to presence
of the essential oils that have limited cytotoxicity
against colon carcinoma [87]. While the aqueous
extract showed anticancer activity with IC50 33.9
range of the phytochemical constituents that can
inhibit the process of carcinogenesis effectively
and prevent the development of invasive cancer
[88, 89]. As suggested byAbdelhady and Badr
[83], incorporation of Ag-NPs into the different
extracts increased the cytotoxicity against growth
of human colon cancer cells compared to the
crude ones. This was manifested by their reduced
IC50 for different used cell lines. This attributed
to enhancement of total polyphenolic compounds,
the total antioxidant capacity, iron reducing
TABLE C. tiglium
Solvent
Total antioxidant
Total
Seeds 0.570 ± 0.014 487.18 ± 9.40 7.25 ± 0.91 22.521 62 % 36.3
Ethanol
Before 0.444 ± 0.002 617.87 ± 3.85 3.30 ± 0.42 21.205 53 % 36.3
After 0.60 ± 0.04 873.54 ± 9.31 7.06 ± 0.88 29.45 71 % 18.75
P. ether
Before 0.217 ± 0.009 388.24 ± 6.47 0.37 ± 0.08 0.838 22.2 % 50
After 0.36 ± 0.02 485.91 ± 5.18 4.31 ± 0.54 1.25 35.7 % 23
Aqueous
Before 41.525* 33.9*
After 62.74* 3.13*
*: The most effective extract as compared to the others, Value expressed as mean ± SE of four replicates
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WAEL MAHMOUD ABOULTHANA et al.
power and free radicals scavenging activity and
hence increasing the anticancer activity through
lowering growth of the cancer cells.
Incorporation of Ag-NPs into the extract
caused no toxicity in the experimental animals
when administrated orally by stomach tube. This
was in accordance with the concept reported
by Mohanpuria et al. [90] who suggested that
green route synthesis of Ag-NPs found with less
emphasized that the LD50 of the ethanolic seeds
extract-Ag nanocomposite was about7.95 ml/
Kgandthe therapeutic dose was about 0.8 ml/
Kg. In the petroleum ether seeds extract-Ag
nanocomposite, the LD50 was about 5.2 ml/Kg
and hence the therapeutic dose was 0.52 ml/Kg.
Moreover, the aqueous C. tiglium seeds extract-
Ag nanocomposite was found with LD50 about 65
ml/Kg and hence the therapeutic dose was about
6.5 ml/Kg.
In this study, it was concluded that C. tiglium
seeds contain various active phytoconstituents
compounds) and proteins. Incorporation of
Ag-NPs into the different extracts (ethanolic,
petroleum ether and aqueous seeds extracts)
enhanced the antioxidant properties through
increasing the total antioxidant capacity, total
reducing power and free radical scavenging
activity in comparison with the crude extracts.
Therefore, it was shown that the nanoparticulated
extracts exhibited elevated cytotoxicity against
growth of human colon cancer cells compared to
the crude ones. No toxicity occurred as a result
of incorporation of Ag-NPs into the extract when
administrated orally by the experimental animals.
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(Received 29/8/2018;
accepted 3/10/2018)
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