沙漠研究 24-1, 145-152 (2014)
Ἃ₍◊✲ 24-1, 145 -152 (2014)
Journal of Arid Land Studies
Journal of Arid Land Studies－ DT11 Refereed Paper －㸫DT11 Refereed Paper㸫Potent Anticancer and Antioxidant Activities of Active Ingredients Separated
from Solanum nigrum and Cassia italica Extracts
Ahmed M. ABOUL-ENEIN*1), Faten Abu EL-ELA1), Emad SHALABY1) and Hany EL-SHEMY1)
Abstract: The present work discusses the separation and purification of bioactive compounds from the crude ethanolic extracts ofCassia italica and Solanum nigrum leaves. Identification of compounds in the crude extract was accomplished using different
spectrophotometric analyses (MS, NMR and IR). Eight active compounds were identified from crude extract of S. nigrum while six
were identified from Cassia italica. Each active compound was further evaluated for anticancer properties using Ehrlich ascites
carcinoma cell (EACC) line and Hepatoma cell (HepG2) line. The antioxidant activity of each active compound was determinedusing the 2, 2 diphenyl dipicryl hydrazine (DPPH) method. The identified compounds showed variable antioxidant activities. It isapparent from our study that effective drugs produced from Solanum nigrum and Cassia italica tend to support the use of these plants
in the treatment of cancer.
Key Words: Apoptosis, Biological activities, Cassis italic, Pure compounds, Solanum nigrum
italica and Solanum nigrum leaves which showed antioxidant
and anticancer agents.

1. Introduction
Medicinal plants have been used by humans for centuries
in folklore medicine (Sermakkani and Thangapandian, 2012).
Medicinal plants are also incorporated into the historical
medicine of virtually all human cultures. The plants are a richsource of secondary metabolites with interesting biologicalactivities. Therefore, these secondary metabolites have an
important source with a variety of structural arrangements andproperties (Vickers, 2002; El-Shemy et al., 2003, 2007).
Solanum nigrum (black nightshade) is known as a natural
medicinal plant. Solanaceae is well known and has been
screened by researchers for their medical actions. The plants
were extensively used to treat severe diseases such as pain,
inflammation and fever (Acharya and Pokhrel, 2006). As
well, plants were used as an antitumorigenic, antioxidant,
anti-inflammatory, hepatoprotective, diuretic, and antipyretic
agent (Aboul-Enein et al., 2012; Zakaria et al., 2006). As
stated by Desphande and Bhalsing (2013): “an extensive
survey of literature revealed that Cassia is an important sourceof many pharmacologically and medicinally importantchemicals (Skehan et al., 1990), several studies have claimed
the use of some species of Cassia for the treatment of various
diseases but still the pharmacological potential of the otherplants species of the genus are required to be explored(Mazumder et al., 2009; Riboli and Norat, 2003).”
This work aimed to study the influence of isolated andidentified pure compounds from ethanolic extracts of Cassia* Corresponding Author: aboul_enein1@yahoo.com
Giza, Egypt
1) Biochemistry Department, Faculty of Agriculture, Cairo University

2. Materials and Methods
2.1. Collection of plant materials
Wild growing Solanum nigrum and Cassia italica werecollected during February - March, 2012 from El-Alaemeen-Coastel area, Sedi Abd El-Rahman, north-west of Egypt.
Whole plant samples were air dried and leaves were ground toa powder using a mechanical mortar.
2.2. Preparation of extracts
Ethanolic extracts of S. nigrum and Cassia italica wereprepared according to Ferrigni et al. (1982). Fifty grams ofground leaves were extracted with 70% ethanol. The
supernatant was separated and the solvent was removed. All
the extracted materials were used to test for antioxidant andanticancer activities. The remainder of the plant materials
was preserved at -20°C for spectrophotometric analysis.
2.3. Anticancer activity (Cytotoxicity)
2.3.1. Viability of Ehrlich Ascites Carcinoma cells (EACC)
A line of Ehrlich Ascites Carcinoma from National Cancer
Institute (NCI) Cairo, Egypt was used. The viability
percentages of tumor cells (2 ×104 cells) after incubation for 2
h with ethanolic extract (100 ȝg/ml) as well as with saline ascontrol were measured by the modified cytotoxic trypan
blue-exclusion technique of Bennett et al. (1976). For all
㸦Received, November 2nd, 2013; Accepted, February 17th, 2014㸧

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examined materials (and control), 10 ȝl of cell suspension was
mixed with 80 ȝl saline and 10 ȝl trypan blue (0.4%) then thenumber of living cells (non-stained) was calculated using ahomocytometer slide by microscope.
2.3.2. HepG2 cell line
The anticancer activity of the crude extract and thefractionated compounds were tested against HepG-2 cells(Vacsera, Egypt) by SRB assay as previously described(Skehan et al., 1990) or by neutral red assay (Retpetto et al.,2008). Exponentially growing cells were collected using
0.25% Trypsin-EDTA and plated in 96-well plates at
1000-2000 cells/well. Cells were exposed to each test
compound for 72 h and subsequently fixed with TCA (10%)
for 1 h at 4ºC. After several washings, cells were exposed to
0.4% SRB solution for 10 min in the dark and subsequently
washed with 1% glacial acetic acid. After drying overnight,
Tris-HCl was used to dissolve the SRB-stained cells and color
intensity was measured at 540 nm.
2.3.3. Caspase activity assay
After centrifugation of the treated cells as previously
described, caspase-3 enzymatic activity was determined using
CasPASE Apoptosis Assay Kit (Geno Technology Inc. St.
Louis MO, USA). Prior to use, caspase kit reagents were firstprepared, followed by lysis of the treated cells (HepG2)
according to a modification of the manufacturer's protocol. In
this study, cells were lysed with a sonicator and caspase-3
enzymatic activity in the lysates was determined as described.
It essential to mention that the level of caspase-3 (CPP32)
enzymatic activity in the cell lysate was directly proportional tothe color reaction read at 405 nm. The inter-treatment data
were compared to ascertain and confirm the effect of ZVADFMK on caspase-3 enzymatic expression.
2.4. Antioxidant activity
The DPPH (2,2-diphenyl-1-picrylhydrazyl) test was carriedout as described by Breitenbucher and Figliozz (2000). One
ml of plant extract (100 ȝg/ml) was mixed with 1ml DPPH
reagent {0.002% (w/v)/ methanol solution}. After incubationfor 30 min in the dark at room temperature, the absorbance wasmeasured at 517 nm.
2.5. Identification of the active compounds
The crude ethanolic extracts of C. italica and S. nigrum
were first fractionated using TLC technique to separate thedifferent fractions using different combinations of petroleum
ether/ethyl acetate solvents (9:1, 8.5:1.5, 8:2, 7:3 and 5:5) as
the mobile phase. The separated fractions (1-10 which havebest effect) were scratched and eluted with the same mobile
phase, filtered, evaporated, weighed and then used in bioassays.
The pure fractions were analyzed by different

spectrophotometric techniques.
2.5.1. Mass spectroscopic (MS) analysis of potent fractions
The potent fractions of Cassia italica and Solanum nigrum
were analyzed by Mass spectrum (MS).
The mass
spectrometer was scanned over the 40 to 500 m/z range with
an ionizing voltage of 70 eV and identification was based onstandard mass library of National Institute of Standards andTechnology (NIST Version 2.0) to detect the possible fraction
structure.
2.5.2. Fourier transformed infra red (FTIR) spectra
A Perkin Elmer (Waltham, Massachusetts, USA) was used
to obtain Fourier transformed infrared (FTIR) spectra (System
2000) and applied for each pure fraction analysis.
2.5.3. Proton magnetic resonance spectra (1H NMR)
The identification of compounds was confirmed bycarrying out 1H-NMR analysis using NMR Joel GIM, EX 270(400 Hz).
2.5.4. Statistical analysis
Data were subjected to an analysis of variance, and themeans were compared using the “Least Significant Difference(LSD)” test at 0.01 levels, as recommended by Snedecor andCochran (1982). Data are presented as mean ± SD.

3. Results and Discussion
The examined wild plants were used for their biologicalevaluation and the obtained results were illustrated anddiscussed.
3.1. Antioxidant activity
The change in absorbance produced by reduced DPPH wasused to evaluate the ability of tested compounds as antioxidant
activity. As shown in Table 1 (for S. nigrum extract), band(2) came in the first rank which gave the highest antioxidant
activity (80.5%), Figure 1. The two bands (3, 4) came in thesecond category which gave antioxidant activity as 65.6 and50.5%, respectively, while the other five bands gave
antioxidant activity less than 50%. The antioxidant activity often bands isolated from of Cassia italica was evaluated andresults showed that two isolated bands (3 and 5) gave
antioxidant activity more than 60% while the four bands (1, 2,
4 and 6) gave activity less than 60% (45.8, 48.7, 49.5 and 56.7,
respectively).
The other bands gave less than 40%
antioxidant activity. The fractions which have higher
biological activities (8 fractions for S. nigrum and 6 for C.
italica, as in Tables 2 and 3) were chosen for identification
using spectroscopic techniques.
3.2. Anticancer activity
Our previous study (Aboul-Enein et al., 2012) showed that

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Table 1. Antioxidant and anticancer activity % of pure compounds
separated from Solanum nigrum.
CompoundNo.
1

Anticancer activity(against EACC)
76

2

74

65.6

3

72.5

50.5

4

71

65.3

5

74

6

68

60
40.8

7
8

60
60

45.6
30.2

LSD

1.85

1.28

Antioxidant activity
80.5

Each value is presented as mean of triplicate treatments.
significantly at p#0.01 according to Duncan’s multiple range test.

Table 3. Anticancer activity of active ingredients (at 50 and 100
ug/ml) against HepG2 cell line and its effect on Caspase
enzyme expression.
50

100

Caspaseenzyme(Pg/ml)

50
9.4

75.3
45.1

3.16
2.82

4

50
55.1

55.6
78.9

2.95
3.61

5

58.7

71.8

3.48

6

63

72.8

7
8

56.8
69

77.6
77.2

2.8
3.56
3.29

9

29.8

79.4

2.78

10

44.3

78.65

11

32.9

59.8

4.04
2.87

12

33.5

66.3

3.91

13

43.9

65.8

3.61

14
LSD

42.65
1.47

69.6
1.96

3.5
0.032

Compoundno.

Concentration (µg/ml)

Solanum nigram
1
2
3

LSD

Fig. 1. Antioxidant activity of crude ethanolic extract of Cassia italicand Solanum nigrum.

Cassia italica

Each value is presented as mean of triplicate treatments. LSD
significantly at p#0.01 according to Duncan’s multiple range test. Data
are expressed as mean 6 S.D.

Table 2. Antioxidant and anticancer activity of compounds
separated from Cassia italica.
CompoundNo.
1

Anticancer activity(against EACC)
55

2

57

34.8

3

59

78.4

4

59

30.2

5

50.6

68.4

6
LSD

68
0.65

56.7
2.65

Antioxidant activity
48.7

Each value is presented as mean of triplicate treatments.
significantly at p#0.01 according to Duncan’s multiple range test.

Fig. 2. Anticancer activity of crude ethanolic extract of Cassia italica
and Solanum nigrum.

LSD

both ethanolic and water extracts of Cassia italica andSolanum nigrum showed anticancer and antioxidant activities
more than 80%. In this work we repeated the anticancer andantioxidant activities of crude extract and the obtained data
were nearly the same (Fig. 2).
The results obtained from this study represent an important
step towards the effective isolation and characterization of theactive principles from these plants and to understand themechanism of cytotoxic activity of these compounds. Data
from Table 2 showed that five bands (fractions 1-5) isolated
from S. nigrum gave anticancer activity against EACC morethan 70% while three bands (fractions 6-8) came in the second
rank which gave anticancer activity more than 50% (68, 60 and
60%, respectively). Two other bands (fractions 9, 10) gave
anticancer activity less than 50% (not given in Table 2).
Concerning Cassia italica, six bands (fractions 1-6) gave
anticancer activity more than 50% while the others gave
anticancer activity less than 50% (not given in Table 3).
In a similar study Patel et al. (2009) found that both thecrude extracts and the isolated components of S. nigrum
possessed antiproliferative activity on various cancer cell lines.
Antiproliferative activities of the crude organic extract and theisolated compounds were studied on tumor cell lines of liver(HepG2), 21, 22 colon (HT29 and HCT-116), 21, 22 breast(MCF-7), 26 and cervical (U1424, 25 and HeLa27). Data inthe present study showed that S. nigrum extract has significant

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cytotoxicity effect on HeLa Cell Line and affect proteases that
activate programmed cell death (Table 3). These results werein agreement with the results obtained by Riboli and Norat(2003) who suggested that an inverse association exists
between consumption of vegetables and fruits and the risk ofhuman cancers at many sites using Epidemiological studies.
The aqueous extract of S. nigrum contains steroidal glycosides
and glycoprotein which exerted an inhibitory effect on cell
growth and colony formation (of prostate, breast and colorectal
cells) as mentioned by Nawab et al. (2011). In addition, otherstudy (Li et al., 2008) showed that aqueous extract inhibits
growth of cervical carcinoma (U14).
Our data revealed that the action of the active principles
separated from the tested plant extracts act as enhancers for
apoptotic pathway. This is clear from the results of caspase
activity. The data showed that the tested compounds
activated the enzyme action (protease) to hydrolyze proteins ofcancer cells and encourage the programmed cell death ofcancer cells.
3.3. Activity of identified compounds from S. nigrum
Spectroscopic analysis of the eight active compounds
separated from TLC analysis of S. nigrum extract in present
study was found in Figure 3. These fractions were 2,3
Dihydroxypropyl elaidate, Naphtho [2,1-b]furan-2(1H)one,decahydro-3a,6,6,9a-tetramethy, 5-Bromosalicylaldehyde,
12-sulfanyldodecanoic acid Usnic acid monoacetate.
Trilinolein, Niclofen and 8-Azabicyclo [3.2.1] octane-2carboxylic
acid,
3-hydroxy-8-methyl,(2-endo,3-exo).
Concerning the fifth compound (Usnic acid monoacetate), thecompound belongs to the phenolic class. The identified
compounds in Figure 3 mostly belong to the phenolic
compounds and showed high potency to cancer cells. In thisconcern, similar identification has been done by Chou et al.
(2011) from T. candida and U. barbata extracts anddetermined using HPLC-UV analysis. These extracts containmajor phenolic compounds as usnic acid (U. barbata) andnorstictic acid (T. candida). The authors mentioned that
Usnic acid has the strongest anticancer activity towards both
FemX (human melanoma) and LS174 (human colon
carcinoma) cell lines.
Much information is available on the reported inhibitory
effects of specific plant phenolic compounds and extracts on
mutagenesis and carcinogenesis.
Concerning the sixth compound, Trilinolein has anticancer
activity against HepG2 cells. This compound has been
identified as one of the active constituents isolated from Panax
notoginseng and used widely in traditional Chinese medicine.
The effect of trilinolein on the growth of non-small cell lungcarcinoma A549 was investigated by Chou et al. (2011) who

Fig. 3. Suggested chemical structures of identified active compounds
isolated from Solanum nigrum.

found “that the exposure of A549 cells to trilinolein resulted in
the growth inhibition and the induction of apoptosis. The
intracellular reactive oxygen species, ROS, seem to play a rolein the trilinolein-induced apoptosis. They noticed that ROS
produced early in the trilinolein treatment. The mostimportant regulators of trilinolein-induced apoptosis are Bcl-2
family and caspase-3, which are associated with cytochrome C
release and dephosphorylation on the Akt signaling pathway.”
The anticancer activity of the methanol extract of Cassia
italica against two types of cancer cell lines, (Hep-G2, Helacell lines) was also detected (El-Shemy et al., 2007).
Scientists have suggested that it appears extremely unlikelythat any one substance in the extract is responsible for all of theassociations seen between plant foods and cancer prevention.
This is due to the great variety of dietary phenolics, including
flavonoids, and the many types of potential mechanisms
reported (Birt et al., 2001; El-Shemy et al., 2007). These
results were in agreement with that given in Table 3 whichshow that the ethanolic and water extracts of Cassia and S.
nigrum inhibited the growth of EACC cells in vitro and haveantioxidant activity.
This appears to involve
apoptosis-induced cell loss and a lowering in the proliferation
rate of cancer cells.
3.4. Activity of identified compounds from Cassia italica
The identification of the six active compounds from Cassia
italica was performed and the results are given in Figure 4.

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Fig. 4. Suggested chemical structures of identified active compounds
isolated from Cassia italica.

The six active compounds identified from Cassia italica
were 5,6-dichloro-2-methyl-1H-benzimidazole, Trans-2methyl-4-n-pentylthiane, Nifedipine, Propionic acid (3,6,7,8tetrahydro-3,7-methano-2,4,6-trimethyl-2H-oxocin-7-yl)
methyl ester, di-p,octyl phenoxy 1,5 naphthalene dicarboxylase
ester, Propionic acid (3,6,7,8-tetrahydro-3,7-methano-2,4,6trimethyl-2H-oxocin-7-yl) methyl ester and Octadecyl
bromoacetate. From the figure, the first compound is 5,6dichloro-2-methyl-1H-benzimidazole and has high anticancer
activity. This is similar to that obtained by Pathak et al.,(2010) who covered the most active benzimidazole derivatives.
These derivatives have shown considerable biological actions
such as antimicrobial, anti-inflammatory, antioxidant,
anticancer, anticonvulsant, antidepressant, anti-leishmanial andradioprotective. Some new benzimidazole-4,7-diones weresynthesized and reported (Gellis et al. (2008) which suggestthat the compounds perform excellent cytotoxic activity
against colon (HT29), breast (T47D) and lung (A549) cancer
cell lines and shown lowest IC50 values (3 ȝM)). Also
antioxidant activity by synthesis of some 6-flouro-5-substituted
benzimidazole (Ateú-Alagöz et al., 2005) showed strong superscavenging effect on superoxide anion at 10-3 M concentration.
Concerning the third compound Nifedipine, it belongs to
1,4-dihydropyridine (1,4-DHP). The most feasible position
for substitution is 4th which exhibit various activities i.e., as thecalcium channel antagonists (David and Triggle, 2007).
Swarnalatha et al. (2011) also found that “the heterocyclicring is the common feature for various pharmacologicalactivities such as anti-inflammatory activity (Bahekar andShinde, 2002), antitubercular activity (Wachter and Davis,
1998), analgesic activity (Gullapalli and Ramarao, 2002),

antihypertensive, antianginal (Breitenbucher and Figliozz,
2000) antitumor (Boer and Gekeler, 1995), antithrombotic(Sunkel et al., 1990). It binds to L-type channel and also
shows action by binding to N-type channel (David and Triggle,
2007).” Nifedipine, a drug used for treatment of hypertension
and angina, exerts its effect by calcium channel blockade andnitric oxide production. Arslan et al. (1985) measure freecytosolic Ca2+, in the two highly differentiated tumor cell lines,
Ehrlich and Yoshida ascites carcinomas, by using a new
lipid-soluble heavy metal chelator TPEN. The authors
revealed that these ascites tumor cell lines have normal
cytosolic free Ca2+. Also, the effects of calcium channel
blockers (CCBs) on apoptosis of cancer cells were investigated.
CCBs verapamil and diltiazem inhibited apoptosis (Ares et al.,
1997; Nagashima and Goto, 2000). On the other hand, insome cases, they were also shown to be apoptosis inducers(Balakumaran et al., 1996). Concerning the fourth compound,previous reports mentioned that naphthalene derivatives havedemonstrated a strong growth inhibitory activity against celllines (Abdelwahab, 2009). Therefore, these derivatives ofnaphthalene might be responsible for the anticancer propertiesof the ethanol extract of Cassia italica against EACC cancer
cells. The anticancer and antioxidant activities of the plant
may be due to the first compound that belongs tobenzimidazole derivatives that have shown considerable
biological actions such as antimicrobial, anti-inflammatory,
anticancer and antioxidant. The enhancing of caspase-3 incancer cells after incubation with anticancer compounds
reveals the apoptosis of these cells.
It is essential to mention that the anticancer and antioxidant
activities are associated with the content of phenolics,
including flavonoids. Our preliminary studies showed a good
relationship between antioxidant potency and the total phenoliccontent as well as between the antioxidant potency and the
reducing power (Abou Elalla and Shalaby, 2009). The total
phenolic contents correlated significantly and positively withthe activity of antioxidation enzymes. In addition, theanticancer activity was found to be related to alkaloids andphenolics from C. italica and S. nigrum. The activity may berelated only to phenolics from water hyacinths or to glycosides
and plant acids from C. italica (Trease and Evans, 1989).
All the extracts which gave high anticancer potency havehigh antioxidant activity while the opposite trend is notobserved (Aboul-Enein et al., 2012; Abou Elalla and Shalaby,2009). In this concern, cancer is a multistage process definedby at least three stages: initiation, promotion, and progression(Ames and Gold, 1992; Schulte-Hermann et al., 1990).
Oxidative stress interacts with all three stages of this process.
During the initiation stage, ROS may produce DNA damage
by introducing gene mutations and structural alterations into

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the DNA. In the promotion stage, ROS can contribute toabnormal gene expression, blockage of cell-to-cellcommunication, and modification of second messenger
systems, thus resulting in an increase in cell proliferation or adecrease in apoptosis of the initiated cell population. Finally,
oxidative stress may also participate in the progression stage ofthe cancer process by adding further DNA alterations to theinitiated cell population (Klaunig et al., 1998).
As a conclusion, the present study showed that crude
extract and the isolated compounds from Cassia italica andSolanum nigrum leaves have anticancer and antioxidant
activities. The results showed that 8 compounds were
purified from ethanolic extracts of Solanum nigrum whereas 6
extracted from Cassia italica which have anticancer andantioxidant activities. There is a good relationship between
the antioxidant potency and reducing power. Treatment of
the cancer cells with the active compounds led to their growth
inhibition and the induction of apoptosis in cancer cells.
Intracellular reactive oxygen species seem to play a role in the
induced apoptosis.
It is apparent from our study that effective drugs produced
from Solanum nigrum and Cassia italica tend to support thetraditional medicinal use of the plants in the treatment of cancer
and could lead to development of local pharmaceuticalindustries, thereby enhancing self-reliance and reducing drug
importation.
Acknowledgements
This work was fully supported by a grant from the Scienceand Technology Development Fund (STDF-project ID:312),
Cairo, Egypt.

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