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April 30, 2018
Archives • 2018 • vol.1 • 75-85
http://pharmacologyonline.silae.it
ISSN: 1820-8620
STUDIES OF THE BIOLOGICAL ACTIVITY OF CASSIA FISTULA
Safwat GM1, Hamed MM2*, Moatamed SA1
1Faculty of Biotechnology, October University for Modern Sciences and Arts, Giza, Egypt.
2Medicinal Chemistry, Theodor Bilharz Research Institute, Giza, Egypt
manalayman90@yahoo.com
Abstract
Cancer has been the most dreadful disease from ages till now. However, with the passage of time it been more
understood its mechanism, types, some causing factors, and way of its cell duplication. By the progress of science
there are now more than one available method for treatment, diagnosis or prevention of cancer. But this
progress did not reach till now cheap, fast and effective specific targeting treatment for all types or even one
type. For this aim, the field of searching for alternative natural compounds extracted from plants to replace those
expensive non effective targeting compounds has been wider and advanced. One of familiar plant families is
Leguminosae specifically class of Cassia fistula plant. This type of plant has been examined to ensure its biological
activity and by detecting and analysis it show anticancer activity against liver cancer cell line; hepatocellular
carcinoma HepG2. C. fistula volatile oil extract was able to inhibit proliferation for HepG2 cancer cell lines at 3.05
0.08µg/ml. The methanolic and oil extracts of Cassia fistula showed good brine shrimp larvicidal activity with
lethality concentration (LC50) of 15 and 55 µg/mL, respectively. Finally using GC-MS, the essential oil compounds
were identified, whereas 39 compounds produces and accounted for 90.46% of the total oil, the major compound
was compound sulfurous acid; cyclohexyl-methyl octadecyl ester (21.62%).
Keywords: Fabaceae, essential oil, HepG2 treatment, Cassia fistula, phytochemical.
PhOL Safwat et al. 76 (pag 75-85)
http://pharmacologyonline.silae.it
ISSN: 1824-8620
Introduction
"Humans have been plagued by diseases throughout
the history of civilization" (Laford et al., 1988). In ancient
time, at lack of research evolution, it was the begging of
asking questions to understand the mutation definition,
causes, cases and how to deal with it. The next
worldwide dreaded disease at that time was cancer what
encourages the United States Senate to assign specific
team from researchers and scientists to conquest cancer
and it was the first financial solved issue for researching
at that time. (Lowe-Kenty et al., 2011).
Nowadays it is more clear the main difference in
definition either in the known structure between
"Cancer" and "Tumor". Generally "cancer" is said for the
whole disease. However, the mass of cells that start to
divide and grow in an abnormal way and the repair
mechanism cannot able to force it to its regular form; it
forms what known scientifically by tumor. The
differentiation character between tumor and cancer is
the ability for this mass of cells to move from its original
or primary part of tissue or organ to another place inside
the body by the aid of circulatory system, what is
scientifically known as "metastases". Any tumor mass has
the option of metastases now it should be called
"Cancer". (Balachandram et al., 2005)
One from cancer types that widely spread is liver
cancer, its tumor where the primary origin forms in liver
tissue. Liver is the largest organ inside our bodies as it
considers the sixth most common cancer worldwide, liver
cancer has more than one type according the type of
cancerous cells, but Hepatocellular Carcinoma is the most
common liver cancer known. Hepatocellular Carcinoma
considers more than 90% of all cancer liver.
While the using traditional methods for treatment
have a lot of side effects and disadvantages;
chemotherapy and clinical drugs have targeting issue as
they do not target only the cancer cell and force it to
apoptosis but it effect all normal cells throughout this
process, radiotherapy also more than side effect as
changing in color of skin due to exposing to high
radiation that sure effect other organs all this in addition
to their high cost.
Using of plants in the new medical and
pharmacological investigation is the alternative way of
treatment to replace those methods (Abdel-Gwad,
2000a; Abdel-Gwad, 2000b). After the wide variety of
existence effective natural compounds that have
significant antioxidant, anticancer and chemo preventive
activity, all studies were directed to report and list all
effective phytocompound with reference cases (Hamed,
2007; Hassanein et al., 2010; Irshad et al., 2010; Hamed,
2015; Hamed, 2017).
One of popular and effective medical family in plant is
The Fabaceae, Fortunately one of effective class that has
anticancer potential and chemo-prevention activities is C.
fistula that rich by magnificent compounds (Basker et al.,
2012). This plant has been experimented for many tests to
ensure its safety on the normal cell that was used in this
study as brine shrimp larva and detect its cytotoxicity
against various cancer cell lines.
Materials and Methods
1. Plant Collections:
The leaves of Cassia fistula family Fabaceae was
collected at October from EL-Orman National Garden and
identified by Prof. Dr. Wafaa Amer, Professor of Plant
Taxonomy, Faculty of Science, Cairo University. Voucher
specimen no. CF 1 has been deposited at the Herbarium
of the Medical Chemistry Department, Theodor Bilharz
Research Institute, Giza, Egypt.
2. Experimental Material and Chemicals:
The used solvents and reagents used in this study were
all analytically graded as; hydrocholoric acid, ammonium
hydroxide, citric acid, salicylic acid, mercuric choloride,
diethyl ether, sodium sulfate, sulfuric acid, chloroform,
acetic anhydrides, ammonium solution, magnesium
powder, sodium hydroxide, ferric chloride, α-naphthol,
and ethanol. Other solvents/reagents used for brine
shrimp assay were saline (Instant Oceanic, Marine land
Labs, USA) and brine shrimp's eggs (Artemia Inc.,
California). Also for MTT assay other chemicals were used
as Dimethyl sulfoxide (DMSO), trypan blue dye (obtained
from Sigma St. Louis, Mo., USA), Fetal Bovine serum,
DMEM, RPMI-1640, HEPES buffer solution, L-glutamine,
gentamycin and 0.25% Trypsin-EDTA (obtained from
Lonza Belgium). Cell lines that used were from the
American Type Culture Collection (ATCC, Rockville, MD)
as it was Hepatocellular carcinoma cell line HepG-2. All
solvents, materials and acids were fetched from Merck
Chemical Company and Sigma-Aldrich Company.
3. Essential Oil GC/MS Analysis:
The used system was a combination between gas
chromatography and mass spectrometry carried out on a
GC/MS System: Thermo Scientific TRACE 1310 Gas
Chromatography attached with ISQLT single quadrupole
Mass Spectrometer detector, under specific conditions:
Column: DBS-MS, 30m; 0.25mm ID (J&W Scientific),
Ionization mode: EL, Ionization voltage: 70ev,
Temperature Program: 40 °C (5 min)-275 °C (5 min) AT 5
°C/min, Detector Temperature: 300 °C, Injector
temperature: 300 °C, Carrier gas: Helium; Flow 1 and
Searched library: WILEY & NIST MASS SPECIRAL DATA
BASE. This work was done at The Regional Center for
Mycology & Biotechnology, Al-Azhar University, Cairo,
Egypt.
4. Extraction procedures:
PhOL Safwat et al. 77 (pag 75-85)
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ISSN: 1824-8620
Leaves of Cassia fistula (1 KG) were crushed, dried and
collected in a container in aim to start extraction process
at room temperature. Extraction process was done with
85% methanol for several times then filtrated by using
Whatman filter paper NO.1.
Evaporation mechanism of filter papers to reach
dryness was done by rotary evaporator in vacuum at 50
ºC to have 30gm of brownish viscous residue. The
resulted methanol extract was tested for variety of
phytochemical screening and availability/cytotoxicity
test using brine shrimp eggs as a normal cell and HepG2
cell lines ( hepatocellular carcinoma cell lines) as a cancer
cells.
4.1. Hydro-Distillation process for oil extraction:
Extraction and manufacture of essential oil is
performed by simple and cheap process called Hydro-
Distillation process. Advantages of using specifically this
process in addition to its low cost but also it consume less
stream, less consumed time, and higher oil yield. In this
stage leaves of Cassia fistula (2 KG) were sliced into small
pieces and collected in the Clevenger apparatus, placed in
boiling water (5 liters). The main purpose of using heat
and boiling water is to break down and burst the cell
structure of the plant that will aid to releasing the
essential oils. In order to collect the resulting oil,
molecule of oil carried out by the stream in a long pipe
channels through a cooling tank to help molecules to
return in its liquid form, the process that last for 4 hours.
By using diethyl ether and since oil is lighter than water
that will appear floating on the surface of water it will be
easy to extract the oil from the mixture (Hamed, 2007).
Subsequently, the collected oil was then sent to
National Cancer institute to examine the cytotoxicity of
the extracted compounds. Moreover it was also sent to
be analyzed with Gas chromatography to identify its
ingredient.
5. Cytotoxicity and Cell viability assays:
In order to detect and determine if the oil extraction is
firstly safe to be used on normal cells and has no side
effect there was a primary check test done using brine
shrimps larva.
After the incubation period IC50 test was measured
using probit analysis by assessing the confidence intervals
to 95%, to identify that inhibitory concentration (IC50) for
all test tubes. Any LC50 less than 100pm was pointed to
be considered as active, while any LC50 less than 1000
µg/mL pointed to be toxic, non-toxic value starting from
any LC50 greater than 1000 µg/ml.
5.1. Brine Shrimp Lethality Assay (BSLA):
By obtaining of brine shrimp eggs they were added to
sea salt water and incubated for eggs hatching in a plastic
container with dark cover to prevent any light passages
to reach the required hatching conditions in addition to
the light normal partial. The eggs were added directly to
the dark side of the plastic container; once those eggs
hatched they will move spontaneously to the light side
that associated with lamp above it. Stage of hatching and
maturation of eggs took 48 hour to be completed with
product known now as larva. 4ml of the pre-prepared
seawater was added to facilitate toxicity/viability
measurements. The mixture solution that contains
seawater was divided into seven test tubes. One tube is
for control testing, the rest of the seven tubes were
measured for toxicity at 3, 50, 30,100, 200, and 400 ppm
in 10ml seawater solution added to methanol (1%). In
order to evaporate the added methanol as it has negative
and harmful effects on the normal cells, each test tube
was well mixed and immersed in the water bath at 60°C
for 5 min. In each test tube 10 larva were added. After
one day of incubation for the uncovered test tube,
number of viable larvae was counted and recorded for
the analysis (the data were analyzed and LC50 values
calculated according (Ipsen and Feigi, 1970; Hamed et al.,
2016).
5.2. Antitumor assays:
The cells were grown on RPMI-1640 medium
supplemented with 10% inactivated fetal calf serum and
50µg/ml gentamycin. The cells were maintained at 37ºC in
a humidified atmosphere with 5% CO2 and were
subcultured two to three times a week.
For antitumor assays, the tumor cell lines were
suspended in medium at concentration 5x104 cell/well in
Corning® 96-well tissue culture plates, then incubated for
24 hr. The tested compounds were then added into 96-
well plates (three replicates) to achieve twelve
concentrations for each compound. Six vehicle controls
with media or 0.5 % DMSO were run for each 96 well plate
as a control. After incubating for 24 h, the numbers of
viable cells were determined by the MTT test. Briefly, the
media was removed from the 96 well plate and replaced
with 100 µl of fresh culture RPMI 1640 medium without
phenol red then 10 µl of the 12 mM MTT stock solution (5
mg of MTT in 1 mL of PBS) to each well including the
untreated controls. The 96 well plates were then
incubated at 37°C and 5% CO2 for 4 hours. An 85 µl aliquot
of the media was removed from the wells, and 50 µl of
DMSO was added to each well and mixed thoroughly
with the pipette and incubated at 37°C for 10 min. Then,
the optical density was measured at 590 nm with the
microplate reader (SunRise, TECAN, Inc, USA) to
determine the number of viable cells and the percentage
of viability was calculated as [(ODt/ODc)]x100% where
ODt is the mean optical density of wells treated with the
tested sample and ODc is the mean optical density of
untreated cells. The relation between surviving cells and
drug concentration is
plotted to get the survival curve of
each tumor cell line after treatment with the
specified
compound
.
The 50% inhibitory concentration (IC50), the
PhOL Safwat et al. 78 (pag 75-85)
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ISSN: 1824-8620
concentration required to cause toxic effects in 50% of
intact cells, was estimated from graphic plots of the dose
response curve for each conc. using Graphpad Prism
software (San Diego, CA. USA) (Mosmann, 1983;
Hassanein et al., 2010).
6. Preliminary phytochemical screening for C. fistula:
The dry powder and extracts of C. fistula leaves were
subjected separately to the following phytochemical
tests.
6.1. Test for saponins:
10gm from the leaves extract was added to distilled
water. This mix was strongly shaken then left for 5
minutes to know if this plant contains saponins or not.
Appearance of white bubbles after left period have been
finished was an indicator for the presence of saponins in
the plant (Harbone, 1973).
6.2. Test for carbohydrates and glycosides:
1 gm of extraction was mixed with 10ml of 50% ethanol
in test tube, then the mixed ethanol solution transferred
into another tube with 0.5 ml of ethanolic α-naphthol
solution, a drops of 1ml sulfuric acid was added to the
wall of the test tube to appear violent ring, appearing of
this ring is a clear indicator for the presence of
carbohydrates and glycosides in the plant (Sofowora,
1993).
6.3. Test for sterols and Triterpenes:
After mixing 10gm of leave extract with 20 ml of
chloroform, some drops of sulfuric acid was added on the
wall of test tube. Appearing of red ring is a positive
indicator for presence of triterpenes.
6.3.1. Salkowski test
5 ml of the chloroform solution and an equal amount
of sulphuric acid were added carefully on the wall of test
tube. Appearing of a red color is a positive indicator of
sterols and /or triterpenes.
6.3.2. Liebermann-Burchard test
Evaporate about 5 ml of the chloroform solution of
extract to a small volume then added to 1 ml of acetic
anhydride and some drops of sulfuric acid about 2 ml
were added on the wall of test tube. Reddish brown ring
at the junction between the two layers is a positive
indicator for the presence of unsaturated sterols and/or
triterpenes (Shmidt, 1964).
7. Test for Alkaloids:
By adding 100ml of dilutes HCl on 10gm of leave
extract mixed with ammonia solution (needed for
naturalization) that followed by extraction using
chloroform. The extract was filtrated and evaporated till
it become dry, then dissolved in 2ml of HCl. It supposed
to show slight precipitate in the bottom. Disappearing for
this white precipitate is an also indicator that the plant
doesn't contain alkaloids (Shellard, 1957).
8. Test for Tannins:
10gm of leaves extract was added to 20ml of 50%
ethanol and ferric chloride solution. Green blue color
appearance was the indicator for existence of tannins in
the leaves (Gonzalez and Delgado, 1962).
9. Test for flavonoids:
150ml of 1% HCl was added to 5gm of leaves extract
followed by adding of magnesium metal powder.
Formation of a red color was a positive indicator to
presence of flavonoids (Marby et al., 1970).
Statistical analysis
The data was subjected to analysis of variance
(ANOVA), SPSS (Version 17). Statistical analysis between
groups was performed using 1- way ANOVA. The analysis
was based on at least three replications of every
experiment that produced quantitative data.
Results
Preliminary phytochemical screening for C. fistula
revealed the presence of oils, saponins, carbohydrates
and/or glycosides, sterols and/or triterpenes, tannins,
flavonoids and absence of alkaloids (Table 1). The
evaluation for cytotoxicity was applied in vitro and the
observed results from the methanol extract detect the
presence of a huge amount of bioactive compounds. The
extract was examined using MTT assay (3-(4,5-
Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide).
However, to determine the activity for the oil extract
from the C. fistula plant the extract was send to the
National Cancer Institute to test either this oil has
anticancer activity or not. And after observation of the oil
on more than one type of cell lines, oil extraction of C.
fistula has shown a significant effect on hepatocellular
carcinoma cell line (HepG2) (Hamed et al., 2010).
After observation, the oil extract was able to inhibit
proliferation of HepG2 cancer cell lines at (3.05
0.08µg/ml) compared to Doxorubicin which exhibited
antitumor activity at (4 µg/ml) as a control (Fig.1) (Hamed
et al., 2010). According to the guidelines of the American
National Cancer Institute (NCI) stated that the activity
limit for extract at 50% inhibition time (IC50) of cell
proliferation after 72 hours should be less than 30 µg/ml.
However, when the IC50 of the extract is less than 20
µg/ml it considers having highly level of cytotoxicity
(fig.1). In this study, the results showed a cytotoxic effect
of oil extract of C. fistula on HepG2 cancer cell lines.
Although the results also showed IC50 value lower than
that stated and specified by NCI to determine it as
anticancer agents.
The observed lethality of C. fistula extracts to brine
shrimps indicated the presence of potent cytotoxic and
probably antitumor components of this plant. According
to Meyer et al., crude plant extract is active if it has an
LC50 value of less than 1000 µg/ml while inactive if it is
greater than 1000 µg/ml (Meyer et al., 1982; Gupta et al.,
1996).
PhOL Safwat et al. 79 (pag 75-85)
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ISSN: 1824-8620
The degree of lethality was directly proportional to the
concentration of the extract. Maximum mortalities (100%)
were observed at a concentration of 400 µg/mL in both
extracts. Based on the results, the brine shrimp lethality
of C. fistula extracts were found to be concentration-
dependent. The observed lethality of C. fistula extracts to
brine shrimps indicated the presence of potent cytotoxic
and probably antitumor components of this plant. The
methanolic and oil extracts of Cassia fistula tested
showed good brine shrimp larvicidal activity with lethality
concentration (LC50) of 15 and 55 µg/mL, respectively
(Fig.2 and 3). Analyses of the essential oils from the
leaves of C. fistula were carried out using hydrodistillation
method, (Table 2) shows representative data for the
major constituents in its leaf essential oils, these being
identified using GC-MS. Accordingly, 39 compounds were
identified and accounted for 90.46% of the total. Of the
mixture of alkaloid, sesquiterpenelactone, monoterpene,
pseudoguaianolide, stearaldehyde, steroid, flavonoids,
alcohols, fatty acid, alkenes and alkanes, the main
constituents present were the sulfur containing
compound sulfurous acid, cyclohexyl-methyl octadecyl
ester (21.62%), 3-(2,2- dimethylpropylid-
ene)bicyclo[3.3.1]nonane-2,4-dione (20.72%), 2,6-di(t-
butyl)-4-hydroxy-4-methyl-2,5-cyclohexadien-1-one
(10.13%), hexadecanoic acid (CAS) (3.99%) and
pivaloylacetone, enol (2.96%). The remaining constituents
of leaf oils were, octadecanoic acid, 3-hydroxy-2-
tetradecyl-, methyl ester (CAS) (2.65%), 2,5-
cyclohexadiene-1,4-dione, 2,6-bis(1,1 dimethyleth-yl)-
(2.62%), l-(+)-Ascorbic acid 2,6-dihexadecanoate (2.63%),
Cholestan-3-one, cyclic 1,2-ethanediyl acetal, (5à)- (CAS)
(0.48%). Interestingly, although the alkaloid compounds,
Ibogamine-18-carboxylic acid, 16,17-didehydro-9,17-
dihydro-9, 20-dihydroxy-12-methoxy-, methyl ester (20S)-
and dichotine, 19-hydroxy-11-methoxy-, 2-acetate were
present in C. fistula leaf tissue in percent of (0.62%) and
(0.25%), respectively also, flavonoid compounds;
quercetin-7,3',4'-trimethoxy (0.23%), and Lucenin 2
(0.36%) were detected (Fig. 4), these perhaps being an
indicator of a molecular marker for C. fistula biological
importance. This IC50 value was observed to have effect
anticancer activity on HepG2 liver cancer cell lines to be
considers as a new promising anticancer potential agent.
This result is suggested to be due to presence of specific
phytochemicals in the extract of Cassia fistula, as there
was previous study showed that presence of flavonoids
in the extract causes inhabitation for liver cancer cells.
From those several compound are; phenolic acids that is
expermintally known for anticancer potential by causing
apoptosis in tumor cells as a result for blocking the DNA
synthesis in the cell (Morris 1999; Yuenyongsawad et al.
2013), lectin that found in legumes as a phytoconstituent
has the ability to bind with glycoconjugates which
present specifically on tumor cells, saponin's anticancer
activity is dynamic by interfering with the replication of
DNA, and as a result it prevents sequence the
proliferation of cancer cells that leads to tension
decrease of the aqueous solution that known as
lyobipolar property (Isil et al., 2015), and tannins are
another phytochemical compound which is found
affluently in C. fistula that have the antioxidant activity
and cancer-prevention activity. These activities could be
performed by tannins as they are able to bind to the
former of metallic ions and protein of the cells that force
it to apoptosis cycle. N-hexadecanoic acid and it showed
significant cytotoxicity against human colorectal
carcinoma cells (HCT-116) with an IC50 value of 0.8 µg/mL
(Ravi and Krishnan, 2017). Baader et al., reported that;
ascorbic acid (AA) was found to be cytotoxic to
neuroblastoma cells in vitro and in vivo (Baader et al.,
1994). Steroids are biologically active constituents, they
are considered to be a part of plants’ defense systems,
and as such have been included in a large group of
protective phyto-compounds found in plants named
phytoprotectants (Morrissey and Osbourn 1999; Gus-
Mayer et al., 1994).
Steroids play critical roles in a number of disorders,
including malignancies like prostate cancer, where
steroid production inside and outside the tumour
promotes cancer cell aggressiveness (Lubik et al., 2016).
Moreover, several alkaloids exhibit significant
biological activities, such as the relieving anticancer
effects (Li et al., 2007). Hamed et al., says; a flavonoid
compound quercetin exhibited inhibitory activity against
colon carcinoma cells (HCT-116) with IC50 value of, 10
µg/ml (Hamed et al., 2015).
Finally we strongly can attribute the potent cytotoxic
activity of C. fistula essential oil to its constituents from
alkaloids, sesquiterpene-lactone, steroid and flavonoids.
Taken together, this data revealed a potential use for
the C. fistula oils for medicinal applications.
Acknowledgments
This research was supported by Vice Dean of
biotechnology faculty at October University for Modern
Science and Arts (MSA) Dr. Gehan Safwat and Prof. Dr.
Manal Mortady Hamed, Professor of Medicinal Chemistry,
Medicinal Chemistry Department at Theodor Bilharz
Research Institute.
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assay for cellular growth and survival: application
to proliferation and cytotoxicity assays. J
Immunol Methods. 65: 55-63.
25. Ravi L, Krishnan K. (2017). Cytotoxic
potential of N-hexadecanoic acid extracted from
Kigelia pinnata leaves. Asian J Cell Biol. 12 (1): 20-
27.
26. Shellard EJ. (1957). "Practical Plant
Chemistry" Pitman Medicinal Publishing Co., LTD,
London. 53: 54.
27. Shmidt J. (1964). "Organic Chemistry"
Oliver and Bayed, Edinburgh and London, 8th Ed.
318-673.
28. Sofowora A. (1993). Medicinal Plants and
Traditional Medicinal in Africa. 2nd Ed. Sunshine
House, Ibadan, Nigeria: Spectrum Books Ltd;
Screening Plants for Bioactive Agents; 134-156.
29. Yuenyongsawad S. (2013). Anti-cancer
activity of compounds from Bauhinia
strychnifolia stem. Retrieved from:
http://www.sciencedirect.com/science/article/pii/
S0378874113006727?via%3Dihub.
PhOL Safwat et al. 82 (pag 75-85)
http://pharmacologyonline.silae.it
ISSN: 1824-8620
Table 1: Phytochemical screening of Cassia fistula leaves.
Test Name:
Cassia fistula
Test for volatile oil
Positive
Saponins
Positive
Carbohydrates and/or
glycosides
Positive
Sterols and/or
Triterpenes
Positive
Alkaloids and/or
nitrogenous bases
Negative
Tannins
Positive
Flavonoids
Positive
Table 2: Results of GC/MS analyses of Cassia fistula leaves.
Peak No.
Rt
Area %
M.F.
Identified Compounds
1
8.97
0.7
C6H12O
3-Hexen-1-ol, (E)-
2
12.93
1.05
C8H24O4Si4
Cyclotetrasiloxane, octamethyl-
3
13.02
2.96
C8H14O2
Pivaloylacetone, enol
4
19.55
0.26
C9H9NO
2,6-Dimethylphenyl isocyanate
5
21.83
1.7
C18H34O2
9-Octadecenoic acid (Z)- (CAS)
6
23.14
1.38
C14H30O
1-Tetradecanol (CAS)
7
24.56
1.8
C9H18O2
Nonanoic acid (CAS)
8
26.37
1.03
C22H30N2O3
Aspidospermidin-17-ol,
1-acetyl-16-methoxy- (CAS)
9
26.78
0.56
C12H24N2O3
Pent-4-enoic acid, 2-(2-hydroxy-3-isobutoxypropy
l)-, hydrazide
10
27.57
0.95
C15H24O
4,6-di-tert-Butyl-m-cresol
11
27.91
2.62
C14H20O2
2,5-Cyclohexadiene-1,4-dione, 2,6-bis(1,1
dimethylethyl)-
12
28.05
0.78
C15H24O2
Baimuxinal
13
28.38
10.13
C15H24O2
2,6-di(t-butyl)-4-hydroxy-4-methyl-2,5-cyclohexadien-
1-one
14
29.20
20.72
C14H20O2
3-(2,2- dimethylpropylid-ene)bicyclo[3.3.1]nonane-2,4-
dione
15
30.14
0.65
C15H22O2
1-methyl-6-(1-oxoethyl)-3-oxo-4-prop-2-ylidenecyclo-
nonene
16
33.41
21.62
C25H50O3S
Sulfurous acid, cyclohexyl-methyl octadecyl ester
17
34.05
0.37
C10H13NO3
L-Serine, O-(phenylmethyl)-(CAS)
18
34.28
0.35
C19H26O6
Isochiapin B
19
35.21
0.77
C13H14O4
5-Acetyl-6-formyl-7 aceto-xymethylenecyclo
[2.2.1]hept-2-ene
PhOL Safwat et al. 83 (pag 75-85)
http://pharmacologyonline.silae.it
ISSN: 1824-8620
20
35.72
0.28
C18H30D6O
2,2,3,3,4,4 Hexadeutero
octadecanal
21
36.17
0.23
C18H16O7
Quercetin-7,3',4'-trimethoxy
22
36.26
0.26
C12H10FN5
1H-Purin-6-amine,
[(2-fluorophenyl)methyl]-
(CAS)
23
36.63
0.7
C18H36O
2-Pentadecanone,
6,10,14-trimethyl- (CAS)
24
37.25
1.06
C16H22O4
1,2-Benzenedicarboxylic acid, bis(2-methylpropyl)
ester
25
37.35
0.48
C29H50O2
Cholestan-3-one, cyclic
1,2-ethanediyl acetal, (5à)-
(CAS)
26
37.55
0.46
C18H34D2O
2,2-Dideutero octadecanal
27
38.28
2.65
C33H66O3
Octadecanoic acid,
3-hydroxy-2-tetradecyl-,
methyl ester (CAS)
28
38.38
2.94
C17H34O2
Hexadecanoic acid, methyl
ester (CAS)
29
39.00
0.17
C21H36
14-á-H-Pregna
30
39.19
0.79
C22H23NO6S
2-Demethylthiocolchicine
Formate
31
39.95
0.96
C19H38O4
Hexadecanoic acid,
2,3-dihydroxypropyl ester
(CAS)
32
40.10
2.63
C38H68O8
l-(+)-Ascorbic acid
2,6-dihexadecanoate
33
40.36
3.99
C16H32O2
Hexadecanoic acid (CAS)
34
42.23
1.36
C19H38O2
Methyl stearate
35
43.25
0.50
C35H68O5
Hexadecanoic acid,
1-(hydroxymethyl)-1,2-ethanediyl ester (CAS)
36
43.57
0.49
C24H42O7
L-Ascorbic acid,
6-octadecanoate
37
48.44
0.62
C22H28N2O5
Ibogamine-18-carboxylic acid, 16,17-didehydro-9,17-
dihydro-9, 20-dihydroxy-12-methoxy-, methyl ester,
(20S)-
38
49.16
0.36
C27H30O16
Lucenin 2
39
50.00
0.25
C25H30N2O9
Dichotine, 19-hydroxy-11-methoxy-, 2-acetate
Total %
90.46%
Fig.1: The cytotoxic activity of C. fistula against HepG2 liver cancer.
HepG-2 1
0
20
40
60
80
100
120
200.00
100.00
50.00
25.00
12.50
6.25
3.13
1.56
0.78
0.39
0.20
0.10
0.00
Concentration (µg/ml)
Cell Viability %
PhOL Safwat et al. 84 (pag 75-85)
http://pharmacologyonline.silae.it
ISSN: 1824-8620
Fig.2: Brine shrimp lethality assay of C. fistula methanol extract.
Fig.3: Brine shrimp lethality assay of C. fistula oil extract.
1 3 11 13
14 16
21 25 27
0
10
20
30
40
50
60
70
80
90
100
110
0 100 200 300 400 500
Mortality (%)
Concentration µg/ml
0
10
20
30
40
50
60
70
80
90
100
110
0 100 200 300 400 500
Mortality (%)
Concentration µg/ml
PhOL Safwat et al. 85 (pag 75-85)
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ISSN: 1824-8620
32 33
37 38 39
Fig. 4: C. fistula oil compounds structure.
