沙漠研究 22-1, 107 -110 (2012 )"ICAL 1 / DT X"

Journal of Arid Land Studies－Refereed Paper－

Efficacy Assessment for Several Natural Products with Potential Cytotoxic
Activity Against Breast and Cervix Cancers
Ali M. MAHMOUD1) and Hany A. EL-SHEMY*1)
Abstract: Over more than five decades, cancer remains a national and international health problem regardless of the discovery ofseveral dozens of novel anticancer drugs (natural and synthetic). Accordingly, screening of natural products for promising anti cancer
activity is very initiative field of studies in several countries with diversity of gardenia. Breast and cervix cancers are of the mostcommon gynecological female solid tumors that represent major health problem. Herein, we have assessed the cytotoxic
characteristics of several molecules of natural origin (cerulinin, chrysin, honikiol, limonin, mevinolin, resveratrol, salicin, retinol,ascorbic acid and calciferol) against two different gynecological breast (MCF-7) and Cervix (HeLa) solid tumor cell lines. After
exposure to serial concentrations of the test compounds, SRB-u assay was undertaken and viability assessment was performed via
fitting to Emax model to identify the cytotoxicity parameters such as, IC50 and resistant fraction (R-value). Tested compounds
showed cytotoxic efficacy against both gynecological solid tumor cell lines (MCF-7 and HeLa) with IC50's ranged from 0.61 to 131.1µg/ml. In MCF-7 breast cancer cell line; cerulinin, honikiol, mevinolin and calciferol showed the highest potency with IC50 less than
5 µg/ml. Chrysin showed moderate potency with IC50 of 7.27 µg/ml. Ascorbic acid and resveratrol showed the weakest cytotoxic
activity with IC50 more than 10 µg/ml. In HeLa cervix cancer cell line; cerulinin and mevinolin showed the highest potency withIC50 less than 5 µg/ml. Chrysin, honikiol, resveratrol and calciferol showed moderate potency with IC50 of ranging from 5-10µg/ml. Ascorbic acid was the weakest cytotoxic molecule with IC50 more than 10 µg/ml. Limonine, salicin and retinol failed toexert any cytotoxic effect against MCF-7 or HeLa cancer cell lines in-vitro. RT-PCR analysis revealed that the cytotoxicity of theseproducts is multi-factorial and not solely dependent on p53 expression. Impressively, molecules of potent and moderate potency(except chrysin) showed low resistant fraction in both gynecological solid tumor cancer cell lines. In conclusion, our data showedwide range of variable efficacy of several molecules of natural origin against two hormone dependent solid tumor cell lines.
Key Words: Anti-cancer screening, Breast cancer, Cervix cancer, Cytotoxicity, Natural products

2. Materials and Methods
1. Introduction
Far East, Middle East Sahara, and tropical regions are
considered of the richest terrains of natural products in theworld (El-Shemy et al., 2007). Currently, isolation andpurification of the active fraction or active ingredient amongst
potentially active natural product is getting great scientific andindustrial interests (Shanab et al., 2010).
New compounds for the treatment of neoplasm might be
de novo identified in terms of structure and function(investigational new drug, IND). Also there might be several
well known compounds that recently showed promising
activity against cancer (investigational new action drugs, INA)
(Van Den Berghe et al., 1978). Therefore, the aim of thisstudy is to determine the effect of some natural compounds as
anticancer agent.

2.1 Chemicals and drugs.
Pure natural compounds were purchased from Sigma
Chemical Co. (St. Louis, MO). RPMI-164 media, fetal
bovine serum and other cell culture materials were purchasedfrom AATC (Houston, TX). Other reagents were of thehighest analytical grade.
2.2. Cell culture
Breast cancer cell line (MCF-7) and Cervix cancer cell line(HeLa) were obtained from the Vaccera (Giza, Egypt). Cells
were maintained in RPMI-1640 supplemented with 100µg/mL streptomycin, 100 units/mL penicillin and 10%
heat-inactivated fetal bovine serum in a humidified, 5% (v/v)
CO2 atmosphere at 37ºC (Fig. 1).
2.3. Cytotoxicity assays
The cytotoxicity of crude extract and the fractionatedcompounds were tested against MCF-7 and HeLa cells by* Corresponding Author: helshemy@hotmail.com
1) Faculty of Agriculture Research Park (FARP) and Biochemistry Department, Faculty of Agriculture, Cairo University, 12 613 Giza, Egypt.

Table 1. Effect of natural compounds on the cytotoxicity parameters of HeLa cell line.
CompoundsResveratrol

Cerulinine

chrysinchrysin

Honikiol

Limonene

Mevinolin

Salicin

VIT A

Vit-C

Vit D

IC50

8.0±0.25µg/ml

3.9±0.9µg/ml

6.5µg/ml

6.5µg/ml

5.2±0.71µg/ml--

0.61±0.02µg/ml

--

> 300
µg/ml

33.10±2.75µg/ml

5.7±1.95µg/ml

Resistance
Fraction

3.03±0.4%

0.7±0.4%

45.40%

45.40%

2.47±0.17%>50%

0.33±0.09%

>60%

>100%

0.39±0.1%

0.20±0.10%

P arameter

Table 2. Effect of natural compounds on the cytotoxicity parameters of MCF7 cell line.
Compounds
Parameter

ResveratrolCerulinine

chrysin

Honikiol

M evinolin

VIT A

Vit-C

Vit D -

IC50

19.13±5.9µg/ml

3.13±0.15µg/ml

7.27±2.56µg/ml

4.23±1.4µg/ml

0.67±0.2µg/ml---

30.3±3.2µg/ml

2.62±0.61µg/ml

Resistance
Fraction

0.00±0.00

2.57±2.57%

0.93±0.45%

1.5±0.34%

1.4±1.4%>100%

0.0±0.0%

0.00±0.00%(a) MCF-7 monolayer culture at confluent statecompounds were tested against MCF-7 and HeLa cells bySRB assay as previously described (Skehan et al., 1990).
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 dark place and subsequently washedwith 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 ( Skehan et al., 1990).
2.5. Data analysis
The dose response curve of compounds was analyzed
using Emax model (Eq. 1).
[D]m  + R ……… (Eq. 1)% CR = (100 − R ) × 1 − mm  K d + [D] (b) HeLa monolayer culture at confluent stateFig. 1. Cell Line Image (a)MCF-7 monolayer culture at confluent
state, (b)HeLa monolayer culture at confluent state.

SRB assay as previously described (Skehan et al., 1990).
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 72h 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 dark place and subsequently washedwith 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 ( Skehan et al., 1990).
2.4. Cytotoxicity assays
The cytotoxicity of crude extract and the fractionatedWhere R is the residual unaffected fraction (the resistancefraction), [D] is the drug concentration used, Kd is the drugconcentration that produces a 50% reduction of the maximum
inhibition rate and m is a Hill-type coefficient.
IC50 was defined as the drug concentration required toreduce fluorescence to 50% of that of the control (i.e., Kd =IC50 when R=0 and Emax =100-R) (Al-Abd et al., 2008).

3. Results and Discussion
Cerulinine, vitamin D and Honkiol were showed
moderately active as single agent for the Breast and cervix
cancer. In the other hand, vitamin A and C both showed weak
activity according to IC50 in Tables 1 and 2.
Regarding Limonene and Salicin, no data’s were generated
from both compounds’ (Table 1).
Mevanoline is fairly active as single agent for both cancer
cell lines (MCF7 and Hela; Tables 1 and 2; Fig. 2).
There are national and indigenous rights over plant derived

D-R curve of Honikiol in HeLa cell line
120

100

Survival (%)

80

60

40

2000.01

0.1

1

10100

1000

Conc. (ng/ml)

D-R curve of D3 in MCF-7 cell line
120

100

Survival (%)

80

60

40

2000.010.1

1101001000

Conc. (ug/ml)

D-R curve of Cerulinine in MCF-7 cell line120

screening program was initiated more than three decades ago(Ieven et al., 1979), that identified many antibacterial
antifungal, antiviral, antiparasitic, and other pharmacologically
active substance activities in higher plants (Jang et al., 1997).
For example, Resveratrol has shown as being anti-oxidant,anti-inflammatory and neuro-protective (Li et al, 2009).
To the major health and economic impact of cancer andleukemia, considerable attention has been drawn to thediscovery of cytotoxic agents (Elgendi et al., 2010). The
dramatic side effects of the vast majority of cytotoxic agentsurged scientific research in the direction of modifying
treatment options or treatment modalities (Shaked et al., 2005).
The re-assessment of INA drugs in the field of neoplasia worth
intensive investigation by research teams and us (O'Quigley etal., 2010). Particularly, natural compounds of mild tomoderate adverse effect would represent very rich soil for INA
anti-cancer drug fishing.
Resveratrol (RES) has shown a diversity of beneficial
health effects such as, anti-oxidant, anti-aging, cardio-protective, anti-inflammatory and neuro-protective properties(Li et al., 2009). Due to its anti-oxidant properties, cancer
preventive activities of RES have been studied and proved(Luther et al., 2009). However, the first report mentioning
potential anti-cancer activity of RES was as late as 1997
(Signorelli et al., 2005; Jang et al., 1997). Besides its
anti-leukemic properties (Puissant et al., 2010).

100

4. Conclusion

Survival (%)

8060

In conclusion, the effect of some natural compounds as
anticancer was promising for further clinical studies.

402000.010.1

1101001000

Conc. (ug/ml)

Acknowledgement
This research was fully funded by grants from the National
CFIDS Foundations Inc, Needham, MA 02492-3931, USA.

D-R curve of mevalionin in MCF-7 cell line

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100Survival (%)

8060402000.01

0.11101001000

Conc. (ug/ml)

Fig. 2. The effect of natural compounds on the dose response curve(D-R) of MCF-7 and Hela cell lines.

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