A Study On The Effect of Some Plant Extracts On Certain Malignant Cell Lines in Vitro

Islamic University Gaza
Dean of Higher Education

Faculty of Science
Master of Biological Sciences

A Study on the Effect of Some Plant Extracts on
Certain Malignant Cell Lines in Vitro

Prepared by
Saib H. Al Owini

Supervisors
Dr. Abboud EL Kichaoui
Dr. Basim Ayesh

Submitted in Partial Fulfillment of the Requirements for the degree
of Master of Biological Sciences/Zoology
Department of Biology, Faculty of science

2006
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DEDICATION
Dedicated to my father and my mother
To my wife
To my son Ahmad
To my daughters Shahd, Tasneem and Malak.
To all my family members

v

DECLARATION
I hereby declare that this submission is my own work and that, to
the best of my knowledge and belief, it contains no material
previously published or written by another person nor material
which to a substantial extent has been accepted for the award of
any other degree of the university or other institutes, except where
due acknowledgment has been made in the text.

Signature
Saib H. Al Owini

Copyright
All rights reserved: No part of this work can be copied, translated or stored in
retrieval system, without prior permission of the author.

vi
ABSTRACT
In Palestine like in other countries of the world, cancer is one of the most
serious health problems that affect the duration and quality of the individuals!
life. Enormous efforts are invested to cope with this problem, but unfortunately
limited success has ever been achieved with most of the therapeutic
strategies. These efforts are usually complicated with the need for well
experienced surgeons, lack of specificity and high cost, as well as being
usually accompanied with a wide range of side effects.
As the conventional therapeutic strategies fail to fulfill the major requirements
for a successful cancer therapy, the use of naturally developed anticancer
agents has evolved as an alternative safe, low-cost and convenient one.
Therefore, the use of plant extracts with potential anticancer therapeutic
effects might be particularly significant, especially in Palestine, which is rich in
thousands of plant species known for their medical uses. Moreover, the lack
of expertise, the scares economical resources and the complicated political
situation in Palestine don!t allow the application of sophisticated surgical,
chemo- and radio-therapies to cure cancer.
Therefore, the current study, investigates the effect of crude water extracts
from Bottle gourd (Lagenaria siceraria), Fig (Ficus carica) and Nettle (Urtica
pilulifera) on cell lines derived from different human tissue origins (Hep3b:
Hepatocellular carcinoma; Hela: cervical epithelial cancer; and PC-3: prostate
cancer).
The results showed a concentration-dependent reduction in the final number
of cancer cells in consequence to treatment with the aforementioned crude
extracts. Two kinds of anticancer effects were evaluated and found to
contribute to this reduction: the antiproliferation effect (decreased number of
metabolically active cells) and cytotoxicity (decreased number of live cells).
The three plants examined possess both of the effects with various degrees.
Urtica pilulifera possess the strongest and most profound effects on the three
cell lines, mainly by induction of cell death. On the other hand Lagenaria
siceraria probably affects the three cell lines by a combination of cytotoxicity
vii
and antiproliferation almost to a similar degree. Ficus carica most probably
reduces the final number of metabolically active cells mainly by its
antiproliferative effect.
Both Ficus carica and Lagenaria siceraria are edible plants that were chosen
on the bases of being mentioned in the holy Quran. Therefore, although their
effect is lower than that of Urtica pilulifera their amount in the diet or as a
treatment can be safely scaled up when ingested in their native form. On the
other hand, despite its possible toxicity Urtica pilulifera is frequently orally
used as a medication in many conditions by traditional medicine.
Further studies are needed to assess the active ingredients of Ficus carica,
Lagenaria siceraria and Urtica pilulifera, involved in the antiproliferative or
cytotoxic effects of these plants. These studies must involve the
establishment of in vivo animal models and the application of more efficient
extraction and fractionation techniques.

viii
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Acknowledgment
Praise be to Allah, the lord of the worlds, and peace and
blessings of Allah be upon the noblest of the Prophets and Messengers,
our Prophet Muhammad.
I wish to express my great thanks to Dr. Abboud El-kichaoui,
Associated Professor of Mycology and Director of Biological Science
Master program, the Supervisor of this work, for his kind assistance,
continuous and valuable advices.
I would like also to express my deepest gratitude and
appreciation to Dr. Basim Ayesh Associated Professor of Molecular
Biochemistry, the Supervisor of this work, for initiating and planning this
study, enlightening supervision, useful assistance during the course of
the study.
I delighted to acknowledge for Dr. Maged M. Yassin Associated
Professor of physiology for his kindly efforts and valuable suggestion
during the thesis review.
I would like also to express my sincere thanks and gratitude to my
uncle Shehada M.El-Ebweini Master of science in mother and child
health, for valuable advices and continuous support during the course of
this study.
Finally I would like to thank all my friends and colleagues for their
contributed in some way or another to the present work.

x
CONTENTS
DECLARATION--------------------------------------------------------------------------------
i
COPYRIGHT------------------------------------------------------------------------------------
i
ABSTRACT-------------------------------------------------------------------------------------
ii
ARABIC ABSTRACT-------------------------------------------------------------------------
iv
ACKNOWLEDGMENT--------------------------------------------------------------- v
CONTENTS----------------------------------------------------------------------------- vi
LIST OF FIGURES AND TABLES------------------------------------------------ ix
LIST of ABBREVIATIONS xi
INTRODUCTION 1
I. Overview------------------------------------------------------------------------------- 1
II. Objectives----------------------------------------------------------------------------- 4
LITERATURE REVIEW 5
I. Conventional cancer therapy----------------------------------------------------- 5
II. Plants with anticancer effects---------------------------------------------------- 5
A. Plants mentioned in Islamic literature with therapeutic activity--------------- 6
B. Plants studied in this work---------------------------------------------------- 10
1. Bottle gourde( Lagenaria siceraria ) ---------------------------------- 10
2. Fig (Ficus carica)----------------------------------------------------------- 13
3. Romman nettle (Urtica pilulifera)--------------------------------------- 14
III. Cancer cell lines ------------------------------------------------------------------- 16
A. Hepatocellular carcinoma (Hep3b) cell line------------------------------ 17
B. Cervical epithelial cell line (Hela) ------------------------------------------ 17
C. Prostate cell line (PC-3)------------------------------------------------------ 18
xi
MATERIAL AND METHODS 20
I. Materials ------------------------------------------------------------------------------ 20
II. Methods ------------------------------------------------------------------------------ 21
A. Plant collection------------------------------------------------------------------- 21
1. Romman nettle (Urtica pilulifera)---------------------------------------- 21
2. Bottle gourde( Lagenaria siceraria )----------------------------------- 22
3. Fig (Ficus carica)----------------------------------------------------------- 22
B. Preparation of the crude extract -------------------------------------------- 23
C. Cell culture ----------------------------------------------------------------------- 24
D. Experimental design ---------------------------------------------------------- 25
E. Determination of growth characteristics: -------------------------------- 25
F. Determination of plant extract working concentrations --------------- 25
G. Cell proliferation assay ------------------------------------------------------- 26
H. Trypan blue exclusion viability assay-------------------------------------- 27
I. Determination of morphological changes of the cells in culture ---- 27
J. Statistical analysis -------------------------------------------------------------- 28
RESULTS 29
I. Determination of the cell lines growth characteristics---------------------- 29
II. Determination of the different plant extract working concentrations--- 30
III. Effects of plants extracts on cancer cells proliferation activity in
culture -----------------------------------------------------------------------------------
32
IV. Effect of plant extracts on cancer cell lines viability in culture--------- 39
xii
V. Determination of morphological changes of cell lines in response to
treatment of different plant extracts .----------------------------------------------
45
DISCUSSION--------------------------------------------------------------------------- 51
CONCLUSION ------------------------------------------------------------------------- 63
RECOMMENDATIONS -------------------------------------------------------------- 64
REFERENCES ------------------------------------------------------------------------- 65

xiii
LIST OF FIGURES AND TABLES
Figure 1 Taxonomy of Urtica pilulifera. 21
Figure 2 Taxonomy of Lagenaria siceraria. 22
Figure 3 Taxonomy of Ficus carica. 23
Figure 4 Laboratory instruments used for extract preparations. 24
Figure 5 Growth curves of Hep3b, Hela and PC-3 cell lines.. 29
Figure 6 A preliminary experiment of the effect of Urtica pilulifera, Lagenaria
siceraria and Ficus carica on the viability of Hepatocellular carcinoma
Hep3b cells.
31
Figure 7 Reduction of proliferation activity of Hep3b cells in response to plant
extracts from Urtica pilulifera, Lagenaria siceraria and Ficus carica.
34
Figure 8 Reduction of proliferation activity of Hela cells in response to plant
36
Figure 9

Reduction of proliferation activity of PC-3 cells in response to plant
38
Figure10 Reduction of Hep3b cells viability in response to extracts from Urtica
pilulifera, Lagenaria siceraria and Ficus carica.
40
Figure 11 Reduction of Hela cells viability in response to extracts from Urtica
42
Figure12 Reduction of PC-3 cells viability in response to extracts from Urtica
44
Figure 13 Morphology of Hep3b cells in culture with or without extract. 46
Figure 14 Morphology of Hela cells in culture with or without extract. 48
Figure 15 Morphology of PC-3 cells in culture with or without extract. 50
Figure16 Reduction of viability and proliferation activity of Hep3b (a), Hela (b)
and PC3 (c) cells in response to Treatment with Urtica pilulifera
extract.
55
and PC3 (c) cells in response to Treatment with Lagenaria siceraria
extract.
56
xiv
and PC3 (c) cells in response to Treatment with Ficus carica extract
57
Table 1 Literature summary of studies that examined the therapeutic
potential of plant extracts.
7
Table 2 Literature summary of studies that examined the therapeutic
potential of Islamic plant extracts.
8
Table 3 Summary of calculated concentrations of the three plant extracts
Urtica pilulifera, Lagenaria siceraria and Ficus carica that give 50%
(IC
50
) reduction in proliferation activity of Hep3b, Hela and PC-3
cancer cell lines. The R
2
value for each curve equation is illustrated.
54
Table 4 Summary of calculated concentrations of the three plant extracts
Urtica pilulifera, Lagenaria siceraria and Ficus carica that give 50%
(IC
50
) reduction in the % viability of Hep3b, Hela and PC-3 cancer
cell lines. The R
2
54

xv
LI ST OF ABBREVIATIONS
ATCC American Type Culture Collection
ADA Adenosine deaminase activity
PBS Phosphate Buffer Saline.
BPH Benign Prostate Hyperplasia
DMEM Dulbecco's Modified Eagle's Medium.
EDTA Ethylene-Diamine Tetra-acetic Acid.
ELISA Enzyme-linked Immunosorbent Assay
EGF Epidermal Growth Factor
HCC Hepatocellular Carcinoma.
HPV Human Papilloma Virus.
Hep3b Human Hepatocellular carcinoma cell line.
Hela Human cervical epithelial cell line. .
HL-60 Human, peripheral blood, leukemia, acute myeloid cell line
HepG2 Human hepatocellular liver carcinoma cell line
HepA-H Liver cancer cells.
HEPES 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid. Buffer
hpcps Prostate stromal compartment
IC
50
50% Inhibitory concentration.
Lncap Human, prostate, carcinoma cell line
PC-3 Human prostate cancer cells.
R Coefficient of correlation
R
2
Related coefficient of determination
SE Standard Error of the mean
SHBG Sex hormone binding gluobin
XTT Cell proliferation assays, A colorimetric method based on
the tetrazolium salt
1
INTRODUCTION
I. Overview
Cancer is one of the most serious health problems worldwide, affecting
individuals from different sexes, ages, and races. It is a group of diseases,
characterized by uncontrolled cellular growth with frequent cancer cells invasion
to different body parts and spreading to other organs, a process referred to as
Metastasis. Metastasis is the major cause of cancer related mortality (1). In
2005, cancer was the second leading cause of death among both men and
women and accounted for 13% of the total 58 million deaths worldwide (1). In
2006, about 10.9 million new cancer cases are expected to be diagnosed
worldwide and more than 7.8 million cancer patients may die (1).
According to the latest report of cancer registry unit in Gaza strip, 5500 cases
have been reported over the period from January, 1995 to December, 2003 (2).
In addition, 1026 cancer patients died in 2004 in the Palestinian territories with a
mortality rate of 28.2 per 100.000 (2).
Cancer is also a problem of economical dimensions with a very high level of
expenses associated to it. For example the National Institute of Health, USA
estimates that an overall of $209.9 billion were invested worldwide in 2005, for
the sake of cancer research and management (3).
Cancer is a heterogeneous illness which can originate from many different
organs of the human body. However, the most frequent cancer types in the
world are lung, prostate, stomach, colorectal, and esophagus in men; and breast,
lung, stomach, colorectal and cervical in women (1).
Prostate cancer is the most frequently diagnosed and the second leading cause
of cancer death among men, with 234460 new cases estimated to occur in USA
during 2006, and 27350 American men will die as a result of this disease(3). In
Palestine, the mortality rate of prostate cancer was 1.4 per 100000 during the
period from January, 1995 to December, 2002 (2). Despite the fact there are
2
several cell types in the prostate, nearly all of the prostate cancers are
adenocarcinoma, originating in the gland cells (3).
Liver cancer ranks as the sixth most common type of cancer world wide (3).
According to the Palestinian ministry of health liver cancer mortality rate was 1.6
per 100000 over the period from January, 1995 to December, 2003 (2). Many
different liver related tumors are identified depending on the type of cells where
they originate, from these types about 83% are hepatocellular carcinoma (HCC)
that begin in the hepatocytes, the main type of liver cells.
Cervical cancer is the most common cause of cancer death among women in
developing countries and the second most common caner in women worldwide
(1). It is caused by a change in the epithelial cells, which line the wall of the
cervix, and the most common risk factor for this type of cancer is the human
Papillomavirus (HPV) (1).
In the last decades there were great advances in the diagnosis of cancer as well
as in the field of molecular oncology. However, the cure rate of most cancers
remains low. Several strategies have been used to cure cancer among which the
most common are surgery, chemotherapy, radiotherapy, and immunotherapy.
Other modern approaches such as hormonal and gene therapy were proposed
by researchers to replace conventional cancer therapy, with variable degrees of
success (3, 4). All of these therapies have undesired side effects, they are
usually not available all the time and they are expensive. For instance, in surgery
the immune system is compromised due to the large amount of cortisole
released subsequent to the surgery, which increase the probability of cancer
relapse (5). Moreover, the current use of chemotherapy is accompanied with
difficult side effects. It inhibits bone marrow stem cells proliferation leading to
immune suppression (5). Radiotherapy which is widely used in the world is also
accompanied by a great deal of side effects. Lymphocytes are most readily
affected by radiation resulting in prolonged T-cell suppression (5). Other side
effects such as, bone necrosis, lung fibrosis, skin devascularization, ulceration,
3
nausea, vomiting, and renal damage are also associated with all types of
conventional therapies.
As the conventional cancer therapies failed to completely fulfill the criteria for a
successful cancer therapy, the use of naturally developed anticancer agents has
evolved as an alternative safe, low-cost and convenient one. Nontoxic
chemoprevention agents from natural resources were proposed by researchers
for this purpose.
Historically, plants with known therapeutic potential have long been used to cure
a wide range of diseases. An example for these drugs is Morphine, which is a
plant product discovered in 1861 as an analgesic agent. Later, Quinine the
active component of Cinchona bark was isolated in 1820 as an effective anti-
malaria drug (5, 6). Our Arabic tradition is particularly rich in medical plants that
have been used by pioneer Arabic physicians to establish the basis for modern
therapies. These were also recommended by our Profit Mohammad ( !"# $% &"' ()* + )
and the Holly Quran. Nigella, Garlic, Onion and Fenugreek are famous examples
for these plants that were recently proven to have therapeutic effects on several
illnesses.
The use of potentially curative plants might be particularly significant in the
Palestinian territories where the plain and mountains are covered with more than
2600 plant species of which more than 700 are known for their uses as medicinal
herbs or as botanical pesticides (7).
In this thesis we studied the therapeutic potential of three Arabic and Islamic
traditional plants as anticancer agents. These were Fig (Ficus carica) and Bottle
gourd (Lagenaria siceraria) that were mentioned in the holy Quran in more than
one occasion as paradise and favorite plants and were selected based on that.
Nettle (urtica pilulifera) was also tested as a popular traditional plant used for
healing and diuretic purposes (8).
4
II. Objectives
The aim of the current study is to investigate the effect of Bottle gourd (Lagenaria
siceraria), Fig (Ficus carica) and Nettle (Urtica pilulifera), extracts on Hep3b from
human Hepatocellular carcinoma; Hela: human cervical epithelial cancer; and
PC-3- human prostate cancer cells in vitro on the basis of Arabic and Islamic
traditional medicine.
Specifically this study aims at:
1- Determination of the proliferation activities of each cell line in response to
each plant extract treatment.
2- Determination of percent viability of each cell line in response to each
plant extracts treatment.
3- Determination of any morphological changes of each cell line in each time
performing viability testing assay in parallel.
5
LITERATURE REVIEW
I. Conventional cancer therapy
Despite the latest advances in medical sciences, and progress in strategies of
cancer treatment, cancer currently remains a tragic disease and is one of the
major causes of death worldwide. The principal methods of cancer treatment
include chemotherapy radiotherapy and surgery.
Chemotherapy is a systemic treatment, to which the whole body is exposed.
Among the most successful chemotherapeutic agent are Cisplatin, Mitomycin
and Docetaxel. All of these agents enhance serious side effects or long term
complication (9, 10 and 11). These side effects include kidney damage, hearing
loose, lower blood count, liver damage, nerve damage, and blood vessel damage
(12, 13).
External beams of Radiotherapy are associated with unacceptably high levels of
local-regional toxicity (13). Particularly, it affects the rapidly dividing cells of
mucosa, causing irritative urinary and blood loss (13). Later toxic effects result
from damage to the more slowly proliferating cells such as fibroblasts,
endothelial, or paranchymal stem cells causing chronic fibrosis and vascular
damage (13).
Other undesired side effects, such as, immune suppression, bone necrosis, lung
fibrosis and skin devascularization are seen with all types of conventional
therapies (11, 14).
II. Plants with anticancer effects
Plants are the chief source of natural products that are used in medicine. Even
Aspirin, the world best known and most universally used medication, has its
natural origins from the glycoside salicin which is found in many species of the
plant genera Salix and Populus (15).
6
The scientific literature is rich in epidemiological studies that support significant
differences in the occurrence of cancers between oriental and occidental
populations (16). Generally, populations that consume a high level of natural
herbal products have a reduced incidence of cancer. An example is the low
incidence of colon cancer in Asian countries with high consumption of soybean
products (16). Soy beans are the major dietary source of saponins, which have
been suggested as possible anticancer agents (17).
There is lately a great interest in screening for plants to be used in cancer
prevention and treatment. For this reason, extracts from different plants have
been extensively studied. Table (1) summarizes some of these plants with
confirmed anticancer activity, while the following sections review some of the
studies relevant to this work.
A. Plants mentioned in Islamic literature with therapeutic activity
The use of many types of medicinal plants has been practiced by Arabic and
Moslem physicians long before they were shown to possess any therapeutic
value by modern research. For example, members of the Ginger family
(Zingiberaceae) were mentioned in the Holly Quran in more than occasion and
recently shown to protect mice against experimentally-induced mutagenesis and
tumorigenesis (18). It also induces apoptosis in various immortalized or
malignant cell lines (18). Pomegranate (Punica granatum), is also among the
plants mentioned in Quran as a Paradise plant, and many studies highlighted its
chemo-preventive potential (19, 20). In addition, Nigella (Nigella sativa),
Fenugreek (Trigonella foenum graecum), and Henna (Lawsonia inermis) were
also recommended by the Profit Mohammed (P.B.U.H) as medicinal plants.
Extracts from these plants were recently shown to possess an anticarcinogenic
effect (21). Refer to Table (2) for a summary of these and other studies.
7
Table (1): Literature summary of studies that examined the therapeutic potential of plant extracts
Common
name
Latin name
Type of
extraction
Proposed active
material
Subject Effect Ref.
Neem flowers
Azadirachta Indica
(Meliaceae )
Methanol Crude Rat liver Chemopreventive 22
Turmeric
Curcuma longa
(Zingiberaceae)
Aqueous Gingerol and paradol HL-60 Antiporliferative 23
Green tea
Camellia sinesis
(Theaceae)
Methanol
Epiallocatechin-3-gallat
( EGCG)
T-cell leukemia ATL
Inducing apoptotic
cell death
24
Green tea
Camellia sinesis
(Theaceae)
Aqueous
Epiallocatechin-3-gallat
( EGCG)
Human stomach cancer
cells
Antiproliferative 25
Soybean Glycine max Aqueous Crude
Adenocarcinoma form
colon cancer(HT-29)
Cranberry Vacciniumm acrocarpon Organic Polyphenole
9 cancer cell lines Colon,
oral, prostate
Bamboo Bamboo Methanol Dihydroxypurpurin Colo320, leukemia CMK-7
Inducing apoptotic
cell death
28
Salomn's seal Polygonum tinctorium Organic
Ethyl acetate -and
tryptanthrin crude
F344 rats Chemopreventive 29
Radix Coptidis rhizome Aqueous Crude F344 Chemopreventive 30
Lemon grass Cymbopogon citratus stapf 80% ethanol Crude F344 rats Antimutagenic 31

8
Table (2): Literature summary of studies that examined the therapeutic potential of Islamic plant extracts
Common
name
Latin name
Type of
extraction
Proposed active
material
Subject Effect Ref.
Pomegranate
Punica granatum
(Punicaceae )
- Aqueous,
and organic
pericarp
Polyphenols
Human breast cancer
cell line
Inhibition of
proliferation
32
Pomegranate
Punica granatum
(Punicaceae )
Seed oil Polyphenols Rat skin tumor Chemopreventive 19
Pomegranate
Punica granatum
(Punicaceae )
Seed oil Polyphenols
Mouse mammary organ
culture
Chemopreventive 20
Pomegranate
Punica granatum
(Punicaceae )
Seed oil Linolenic acid Rat colon cancer
Suppress
Azoxymethane
induced cancer
33
Pomegranate
Punica granatum
(Punicaceae )
Aqueous or
oily
compartment
Ellagi, caffeic,
luteolin and punic
acid
Prostat cancer cell line Antiporliferative 34
Garlic
Allium sativum
( Liliaceae )
Oil- soluble
Organosulfur
compounds
Liver cancer Chemopreventive 35
Henna
Lawsonia inermis
( lythraceae)
Chloroform Tocopherol Liver carcinoma cell line Antioxidant 36
9
Henna
Lawsonia inermis
( lythraceae)
Aqueous Crude Rat Hepatocarcinoma
Reduction of
severity
37
Grape
Vitis vinifera
(Vitaceae)
Aqueous Flavanol derivatives
Hepatorenal carcinoma
rat
Chemopreventive 38
Nigella
Nigella sativa
( Ranunculaceae )
Aqueous Crude
Natural Killer cells
activity
Augmentation 21
10
B. Plants studied in this work
1. Bottle gourd (Lagenaria siceraria)
Bottle gourd (Lagenaria siceraria) is one of the cucurbitaceous crops present in
Palestine and other parts of the world. It was mentioned in the Holly Quran as
mercy and therapy for the Profit Yonis ( +,-.%* /,0.% !"#) . It is classified under the
Lagenaria genus of the cucurbitaceae family. The medicinal benefits of
Lagenaria siceraria are rarely dealt with in the scientific debate. In one study the
Lagenin fraction which was extracted from seeds of the Bottle gourd, was
regarded as a novel ribosomeinactivating protein with a ribonucleolytic activity
(39).
The Cucurbitaceae family is composed of thirty four genus members and ninety
three taxa overall. Many members of these genera have been reported as
medicinal plants, and some of these members were considered as anticancer
plants. The Cucurbita L, Ecballium A, Lagenaria Ser, Siraitia, Luffa P, Momordica
L. and Trichosanthes L, are examples for these genera.
The Momordica genus is composed of four species: Momordica charantia,
Momordica balsamina, Momordica dioica and Momordica cochinchinesis. The
Momordica charantia species (Bitter gourd) contains an array of novel and
biologically active phytochemicals, including (triterpenes), proteins and steroids.
Triterpenes in (bitter melon) have been clinically demonstrated to possess the
ability to inhibit the guanylate cyclase enzyme (40). This enzyme is thought to be
linked to the mutagenic signaling of leukemia, and solid tumors (40). Other
phytochemicals that have been documented with cytotoxic activity are a group of
ribosome-inactivating proteins named alpha- and beta - momorcharin, momordin,
and cucurbitacin B. A chemical analog of these proteins was developed (MAP-
30) and reported to be able to inhibit prostate tumor growth (41). The
phytochemical momordin has a clinically demonstrated cytotoxic activity against
Hodgkin!s lymphoma in vivo (42). Several other in vivo studies have
demonstrated the cytotoxic and antitumor activity of the entire plant of bitter
11
melon (43). In one study, a water extract blocked the growth of rat prostate
carcinoma (44). Another study reported that a hot water extract of the entire
plant inhibited the development of mammary tumors in mice (45). Numerous in
vitro studies also demonstrated the anti-cancer activity of bitter melon against
numerous cell lines including liver cancer, human leukemia, melanoma and solid
sarcomas (46, 47).
Momordica cochinchinensis, also known as (Gac fruit) is a bright-red fruit rich in
carotene and lycopene. Similar to Momordica charantia, Momordica
cochinchinensis is postulated to contain some antioxidant components in the fruit
extract. Recently, Gac fruit was found to contain a higher lycopene concentration
than other fruits (48). According to a clinical trial study at the Medical School of
Hanoi University, Vietnam, an oil extract of Gac fruit was found to be effective in
the treatment of liver cancer (49).
In a recent study the ability of Gac fruit water extract to suppress colon cancer
cell line growth in vivo and in vitro was studied. It was found that this extract
inhibited the growth of the colon 26-20 adenocarcinoma cell line, transplanted in
Balb/c mice, reducing the tumor wet weight by 23.6% (49). Moreover, it was
found to inhibit cell proliferation in the (colon 26-20) and (hepG2) cells in a
concentration dependent manner (49).
Cucurbita L. genus is one of the most famous members in cucurbitaceae family,
it is composed of nine species of which are, Cucurbita pepo L (pumpkin),
Cucurbita moschata (crookneck squash ), Cucurbita maxima (winter squash )
and others.
Cucurbita pepo, (pumpkin) is one of the popular plants in Palestine. Its fruits
consist of up to 50% fatty acid, carotenoides, proteins, tocopherol and
phytosterol (50). Pumpkin seeds have been used for a long time in the traditional
medicine in North America and Mexico as an antihelmintic agent and as a
supportive treatment of the bladder disorders and urination difficulties (50). The
childhood enuresis nocturna and irritable bladder have also been treated
12
successfully with pumpkin seeds (50). Pumpkin has also been used to eradicate
tapeworm (50). Furthermore, Pumpkin seeds are considered an alternative
treatment for stage I and II benign prostatic hyperplasia and for irritable bladder
(51). Muscatine is a novel ribosomeinactivating protein that has been recently
purified from pumpkin seeds, and shown to have a strong inhibitory activity to
protein synthesis (52). It has been used for construction of an immunotoxin that
can efficiently and selectively kill cultured human melanoma cells (52).
A novel immunotoxin Muscatine (Ng76) was prepared successfully form pumpkin
extracts and found to efficiently inhibit the growth of targeted (M21) melanoma
cells (53). These results implied that Muscatine could be used as a new potential
anticancer agent. Moreover, the pumpkin seeds extract can modulate the
immunopathological pathways induced by interferon- (50). This finding suggests
an immunoregulatory potential of compounds contained in pumpkin seeds.
The anticancer and antiinflamatory activities of squash (Cucurbita maxima) are
reported by a study carried out on breast, lung, and central nervous system
cancer cell lines (54).
Siraita grosvenorii (Momordica grosvenorii) is the only species in the genus
Siraitia form cucurbitaceae family; its fruits have been used for the treatment of
pharyngitis, and antitussive medicine in Japan (55). Extracts from this fruit also
where shown to possess an anticancer effect. For example they exhibit a
significant inhibitory effect on the two stage mouse-skin carcinogenesis in vivo,
induced by 7,12-dimethyle benz[a]anthracene (DMBA) and 12-0-
tetradecanoylphorbol 13-acetat (TPA) (56, 57).
Trichosanthes Kirilowii (Maximowii) a species of the Trichosanthes genus from
the cucurbitaceae is a popular herbal medicine in China. It has been used to
induce midterm abortion and to treat ectopic pregnancies, hydatidiform and
trophobalstic moles (58). Maximowii has also been found to posses various
pharmacological properties including immunomodulatory, anti- human
immunodeficiency virus, and anti tumor activities (58).
13
Trichosantin is a ribosome inactivating protein produced from the root tubers of
Trichosanthes Kirilowii. This protein has been regarded as an effective anti-tumor
agent that is highly specific to choriocarcinoma cells from trophoplast (59, 60).
The growth inhibitory effect of Maximowii extract on the liver cancer cells (HepA-
H) and the cervical cancer (Hela) cells was investigated in vitro. The data
showed that the Maximowii extract has a strong cytotoxicity and induction of
apoptosis of Hela cells (61, 62 and 63).
Ecballium elaterium L. Rich, Squirting cucumber is one of the most distributed
cucurbitaceous herb in Palestine, locally known as" Qitha al Hamir". In an
ethnobotanical study carried out in Palestine it was reported that "Qitha al Hamir"
is used as a diuretic, and to treat urine!s retention, piles, swollen testicles, and
yellow fever (64). Cucrbitacin B a protein separated from this plant was reported
as an anti-inflammatory and antitumor agent (65).
2. Fig (Ficus carica)
Ficus carica (the common edible fig) is a member of Ficus L. genus which
belongs to the Moraceae family. Many species of this genus such as, Ficus
racemosa, Ficus bengalensis, Ficus awkeotsang Makino, Ficus microcarpa,
Ficus pumila and Ficus carica are of a medicinal value.
For example ethyl acetate extract of Ficus racemosa resulted in significant tumor
growth inhibition by 76% in in-vivo study (66). An extract from Ficus racemosa
was used to contra diet the carcinogenic effect of Ferric nitrilotriacetate a well
known renal carcinogen (67). When rats were orally treated with this extract, they
showed a significant decrease in lipid peroxidation, xanthin oxidation, H
2
O
2

generation, blood urea nitrogen, DNA synthesis and incidence of tumors (67).
These data suggest that Ficus racemosa extract is a potent cancer preventive
agent.
An aqueous extract of Ficus pumila species was reported as an active preventive
agent against cervical cancers in vivo (68). Pectin esterase, an inhibitor of a
14
group of cationic polypeptides, was extracted from Jelly fig (Ficus awkeotsang
Makino). This polypeptide displayed a strong growth inhibition of human
leukemia cell lines via induction of apoptosis in a dose and time dependent
manner (69).
According to an ethnopharmacological study carried out in the United States of
America, Ficus bengalensis has also been reported as a medicinal plant used for
reduce tumor growth on bone (70). In a different study that aimed to demonstrate
the possible antioxidant role of Ficus bengalensis against a cholesterol rich diet,
it was found that treatment of rabbits with a water extract decreased the serum
cholesterol, and triacylglycerol levels (71). In addition, treatment with the same
extracts led to decrease in lipid peroxidation (71).
In an ethnopharmacological survey carried out in Palestine, edible fig was
reported as one of the most popular plants that are used to cure Warts, kidney
stones, respiratory system, asthma and cholesterol (7). A similar study carried
out in Pakistan indicated that edible fig fruits are useful in the case of the head
blood leprosy, nose bleeding, while the root is tonic and useful in leucoderma
and ringworm (72).
An in vitro assessment of the antihilmintic effect of Papaya, Fig and Kiwi against
gastrointestinal nematodes showed that fig and papaya caused marked damage
of the cuticle of Heligmosomoides polygyrus (73).
The antidiabetic effect of Ficus carica leave extracts has been reported. In one
study, the parameters related to oxidative stress in rats were measured and the
results confirmed that the antioxidant status is affected in diabetes, and that
Ficus carica extract tends to normalize it (74).
3. Roman nettle (Urtica pilulifera)
Urtica pilulifera, a facultative wetland plant, is common in many parts of the
world. It is a member of the urticaceae family which is considered as an invasive
plant. Members of the genus Urtica, including Urtica massaica, Urtica parviflora,
15
Urtica pilulifera, Urtica urens, and Urtica dioica are known for the stinging
sensation caused by the injection of histamine, acetylcholine, and Serotonin into
the skin by fine, needle-like projections found on the leaves and stems of the
plant (8, 75). Despite this deterrent, many of these nettle species have been
used for generations in the preparation of herbal medications (76, 77).
Many plants of the family Urticaceae are commonly used in the world for their
anti-inflammatory properties, particularly Urtica leptuphylla. However, Urtica
dioica is by far the most widely used plant of the family, both culturally and
medicinally (78, 79).
Urtica dioica is a popular anti-diabetic drug, acting against hyperglycemia (80).
To determine this role a water soluble extract of Urtica dioica was administered
to rats under oral glucose tolerance test. The results showed that rats receiving
the extract had 33% lower glucose levels than the control (80). Recently, a
number of studies have been conducted to investigate the effect of Urtica dioica
in the treatment of rheumatoid arthritis as well as the mechanisms through which
it acts (81). Urtica dioica is also believed to have an antioxidant role against
oxidative damage (81, 82). Antioxidants halt the free radical chain reaction,
thereby playing a preventive role in cancer, mutations, and accelerated aging.
Oxidative damage is usually caused due to the cellular and tissue destructive
effects of some chemicals usually formed as byproducts of normal metabolism.
These include, superoxide, hydrogen peroxide, hydroxyl radicals, or singled
oxygen all of which are common byproducts of normal metabolism, attack cells
and tissues. An imbalance between active oxygen and antioxidants may cause
stimulation of peroxisomes and polymorphonuclear leukocytes and
macrophages, which can result in diseases such as arthritis and accelerated
aging (81).
In a study on the antioxidant activity of non cultivated plants, it has been shown
that the Urtica dioica extract inhibits lipid peroxidation by more than 50% (82). In
a similar study, it was reported that an aqueous extract of Urtica dioica is the
16
most effective antioxidant among series of some other medicinal plants (83, 84
and 85). Urtica dioica also showed a potential antioxidant effect on ischemic
muscle tissue, generated by a turniquet in rats (86). In another study, antioxidant
activity of Urtica dioica extract was determined to be iron-promoted oxidation of
phospholipids, linoleic acid, and deoxyribose (87). Many of the phytochemicals
shown to exhibit antioxidant activity are polyphenols, such as caffeic malic acid,
common in Urtica extract (88).
Extracts of Urtica dioica were also shown to have an Antiproliferative effects in
vitro. Such effects were determined by testing methanolic aqueous extracts from
deride milled stinging nettles root (89). Lymph node carcinoma of the prostate
cell (Lncap), and human primary culture of the prostate stromal compartment
(hpcps) were cultivated without extract (control) or treated with various extract
concentrations at different time intervals. The cell proliferation was determined
colorimetrically, and the cytotoxicity was tested by trypan-blue dye exclusion test.
The results demonstrated that the methanolic-aqueous extract treated Lncap
cells are reduced in proliferation in comparison to the untreated controls, while
hpcps remained unchanged.
In an ethno-botanical survey carried out in the west bank, Palestine to evaluate
the most popular traditional plants, Urtica pilulifera was reported as a popular
traditional plant used as aphrodisiac, diuretic, and fresh young leaf are eaten to
treat kidney stone Rheumatism and bleeding (64).
Another survey conducted in the west bank and Negev desert, of Palestine
concluded that Urtica pilulifera is a traditional plant widely used in the treatment
of cancer (7). It was suggested that a standard decoction be prepared from 50 g
plant leaves in one liter and taken orally, 150 ml, 3-4 times/day until the condition
improves (7) .
III. Cancer cell lines
A cell line refers to cells derived from a single cell type that have been adapted to
grow continuously in the laboratory and are used in research (90).
17
Prostatic cancer cell line PC-3, cervical epithelial cancer cells (Hela) and
Hepatocellular carcinoma cell line (Hep3b) were used in the study.
A. Hepatocellular carcinoma (Hep3B) cell line
The Hep3b cell line is a hepatocellular carcinoma derived from 8 years human
male juvenile (91). Analysis of the cell culture fluid form Hep3b revealed that 17
of the major human plasma proteins are synthesized and secreted by this cell
line. Also, Hep3b produces the two major polypeptides of Hepatitis B virus
surface antigen. This cell line was used to generate an animal model for
metastatic hepatocellular carcinoma by injection into athymic mouse (101).
The Hep3b cell line was used to determine the cytotoxic effects of 6-xanthone
compounds from the peel of fruits of Garcinia mangostana on hepatocellular
carcinomas using the MTT proliferation assay (102).
A Similar study examined the effect of Ginko biloba extract on cell proliferation
and cytotoxicity in human hepatocellular carcinoma cells using this cell line in
addition to HepG2 cell line (103). In this study cell proliferation and cytotoxicity
were determine by the (MTS) assay.
B. Cervical epithelial cell line (Hela)
The Hela cell line was derived from a cervical epithelial cancer from a 31
years old female, and it has been reported to contain Human Papillomavirus18
(HPV-18) (91). Hela cells have been used in many studies which aimed to
investigate the anticancer role of artificial and natural products.
The antiproliferative effect of Chlorophytum comosum root extract was tested in
vitro against Hela, HL-60, and U937 cells (97). The extract was found to have
antiporliferation effect, and to induce apoptosis in these human cell lines (97).
The cytotoxic effect of the anticancer drug Rhodamine and Herbal extracts of
Glcyrhizan Radix, Rhei Rhizoma and Zingibers Rhizoma on Hela cells was also
suggested (98). Hela cells were used to indicate that the combination of
18
anticancer drugs with some herbal extracts contributes to the enhancement of
clinical outcomes in cancer thereby (98).
In a cytotoxicological study of Iranian conifers on Hela cells, a colorimetric assay
(MTT) was used, and the findings showed that Junperus Sabina extract posses
an inhibitory effect against Hela cells by reducing the cell viability to less than
50%(99). Another similar study investigated the suppressant effects of Vandellia
cordifolia on human cervical cancer Hela cell line. The result indicated that this
plant extract suppressed Hela cell line proliferation by cell cycle progression
inhibition at the G1 to S phase transition (100).
C. Prostate cell line (PC-3)
The Prostate cell line (PC-3) was initiated from a grade-IV bone metastasis
Prostate adenocarcinoma from a 62 years-old Caucasian male (91). The
establishment, characterization, and tumorigenicity of (PC-3) cell line were
reported (92). The cultured cells showed anchorage-independent growth in both
monolayers and in soft agar suspension and can produce subcutaneous tumors
in nude mice.
Electron microscopic studies revealed many features common to neoplastic cells
of epithelial origin including numerous microvilli, junctional complexes, abnormal
nuclei and nucleoli, abnormal mitochondria, annulate lamellae, and lipoidal
bodies. In general, the functional and morphological characteristics of PC-3 are
those of a poorly-differentiated adenocarcinoma (92).
The PC-3 cell line has been used in vivo and in vitro in many studies in the
screening for new anticancer agents. PC-3 cells were used in a recent study to
test the cytotoxic and antiproliferative effects of the crude extract of Thai
medicinal plants for cancer treatment (93). A similar study evaluated the effects
of plant hormones on PC-3 cell line in vitro reported that PC-3 cells exhibited
differential sensitivities to three hormones, and PC-3 proliferation was
significantly inhibited (94). The human prostate carcinoma PC-3 was also used to
determine the chemosensetivity of prostate cancer to genistein isoflavon
19
(metabolites of soy) and -lapachone (plant product) in vitro using trypan-blue
and MTS bioassay (95). The type of cell death, apoptosis or necrosis in response
to treatment of PC-3 cells was determined using different assays such as
annexin V-FITC and PI/TUNEL apoptosis assays (96).
20
MATERIALS AND METHODS
I. Materials
Reagents and disposables
Biological Industries
Biet Haemic, Israel
DMEM powdered medium
0.05% trypsin-EDTA solution
Cell Proliferation kit (XXT kit)
Sartorius.
UK
Sartolab RF/BT Vacuum Filtration Units
(0.2m filter )
Minisart 0.2m syringe filter.
Greiner bio-one.
Germany
Cell culture flasks.
PS Plate six-well.
PS Plate-96-well.
Buffers and solutions
DMEM medium 10% fetal calf serum (inactivated at 55C for
30 min)
25mM HEPES (pH 7.4),
Penicillin (180 units/ml)
Streptomycin (100g/ml)
Amphotericin-B (0.2 g/ml)
HEPES (Buffer) 280 mM NaCl, 1.5 mM Na2HPo4 and 25mM
HEPES
21
II. Methods
A. Plant collection
Three plants were studied in this thesis: Urtica pilulifera, Lagenaria siceraria and
Ficus carica. They were collected at different times, localities and conditions.
1. Roman nettle (Urtica pilulifera)
Urtica pilulifera locally named "Qurrais (t)"), or "Huraiq ( 8 )" ) belong to the
family Urticaceae which is a member of the Urticales order (Figure1). It is an
annual plant that grows to 1 meter high by 0.5 meter wide and lives in many
types of soils, especially light ones, with a PH ranging from acid to alkaline and a
full sun with moderate moisture. This plant withstands frost and is distributed in a
weed of cultivated lands, waste places, hedges and on damp roadsides or near
dwelling. From all of these sites in the middle districts of Gaza, nettle was
collected during March and April, 2005 (the end of winter and the beginning of
spring in Palestine). All of the plant parts including roots, leaves, and seeds were
harvested by drawing the stem from the soil. All plant parts were washed under
tap water and then derided in the shadow for three to five days. Dried leaves and
seeds were grounded by hand and stored in dry and clean bottles until the time
of experiments.

Figure1. Photograph of Urtica pilulifera in its habitat, shot by the
researcher, April 2005.
22
2. Bottle gourd (Lagenaria siceraria)
Figure 2 shows the vigorous annual herb Bottle gourd, locally named "Yaktein
(<E9X)), which is a member of the Cucurbitaceae family. The stems prostrate or
climb up to 5m long, and the leaves are simple, shortly and softly hairy, and non-
aromatic when crushed. Lagenaria siceraria is growing mainly on alluvial sandy
soil and red loam, on flat areas and moderate slopes, on rocky ridges, on river
banks and in dry riverbeds.
Lagenaria siceraria was collected during May and June (beginning of the
summer). Also all parts of the plant (roots, leaves, and fruits) were harvested by
drawing the plant stem. The seeds were isolated form the fruits, then seeds and
leaves were washed under tap water and dried in shadow places for seventh to
ten days. The dried plant parts were grounded by hand and stored in dry and
clean bottles until the time of experiments.
Figure 2. Photograph of Lagenaria siceraria in its habitat, shot by
the researcher, may 2005.
3. Edible fig (Ficus carica)
The edible fig belongs to the family Moraceae (Figure3), centers around the
Mediterranean region, and is commonly cultivated in mildtemperate climates.
Ficus carica is a tree of small dimensions; (3-9m) high with numerous branches
and a trunk rarely more than (17.5cm) in diameter, and contains copious milky
latex. Lateral spread of roots is extensive and, in certain soils, the roots are quit
deep. Plant leaves were harvested in summer 2005, washed under tap water and
23
shadow-derided for a week at least. Deride leaves were grounded and stored in
a dry and clean bottles until the time of experiments.

Figure 3. Photograph of Ficus carica in its habitat, shot by the
researcher, June 2005.
B. Preparation of the crude plant extracts
Twenty grams of grounded dry parts of the upper-indicated parts of each plant
were soaked in 80 ml distilled water (20% dry wt/v). The extraction was carried
out by using a reflux condenser (Figure 4a) at boiling temperature for 30 min.
The condenser returns the extract vapor to the boiling flask. The extract was
cooled at room temperature and filtered using a Buchenr funnel (Figure 4b) with
0.4m cellulose filter paper. Finally the extract was sterilized-filtered using
vacuum filter with 0.2m cellulose filter paper (Sartorius, UK). About 80% of the
extract volume was collected after filtration, and stored in sterilized bottles at 4C
(Stock extract). The different working dilutions were prepared in the cell culture
media as indicated (104, 105, 106, 107 and108).
24
a
b

Figure 4. Laboratory instruments used for extract preparations.
(a) Reflux condenser and (b) Buchenr funnel.

C. Cell culture
The human cervical carcinoma cell line (Hela), hepatocellular carcinoma cell line
(Hep3b) and Prostate cancer cell line PC-3 were obtained from the Hebrew
university of Jerusalem. They were chosen based on there high proliferation
rates and availability. The cell lines were routinely maintained as a monolayer in
Dulbecco's modified Eagle's medium (DMEM) Containing 10% fetal calf serum
(inactivated at 55C for 30 min), 25mM HEPES (pH 7.4), penicillin (180 units/ml),
streptomycin (100g/ml) and amphotericin-B (0.2 g/ml)(4,109). The cells were
grown to confluency in a humidified incubator with 5% CO
2
in polystyrene culture
flasks. They were subcultured by removing the medium and adding 4-6 ml of
0.05% trypsin-EDTA solution. The cells were allowed to detach at (37C) for 5-10
min. About 1/6 of the trypsinized Hep3b or 1/4 of the other cells was passed
twice a week to new flasks containing fresh-medium.
25
D. Experimental design
- In the first (preparatory) experiments, the extracts were prepared from the
desired plant parts. The working extract concentrations were then
determined by testing an array of extract dilutions on one cell type.
- The working extract concentrations were tested for each plant against
each of the three cell lines in terms of cellular proliferation.
- The effects of the same extracts working concentration were further
analyzed by viability assay to determine the type of cellular effect.
- Observation of the morphological changes was carried out in parallel to
the viability assays.
- The data was statistically analyzed and comparison of the results from
different methods was done and reported.
E. Determination of growth characteristics
Cells from Hela, Hep3b and PC-3 cell lines were seeded in 6-well plats, at a
density of 100000 cells/well, as indicated earlier. Three wells from each cell line
were trypsinized and harvested after 24, 48 and 72 hours and the medium was
changed for the cells continuing to grow at each time point. The harvested cells
were counted on hemocytometer and the average number of 3 wells was used
for the growth curve.
F. Determination of plant extract working concentrations
Plant extract-DMEM preparations were prepared by incorporation of sterile stock
extracts into the DMEM, media preparation (the plant extract volume was
included in final volume calculation). The highest extract-DMEM plant
concentration achieved this way was 16% dry wt/v (Stock extract-DMEM), and
the desired extract-DMEM concentrations were prepared by dilution with the
proper volume of complete DMEM medium.
26
To determine the concentration with a 100% cytotoxic effect, 200000 cells were
seeded onto 25cm
2
flasks containing DMEM media for 24 hours. The medium
was then replaced by 8ml of 16, 8, 4 or 0.0% plant extract-DMEM. The flasks
were prepared in triplicates, and the cells were incubated in extract presence for
24 hours. The viability of cells was determined as described in the following
section.
G. Cell Proliferation Assay
A commercially purchased colorimetric kit was used to determin the proliferation
activity of cells (Biological Industries, Biet Haemic, Israel). The method is based
on the ability of metabolically active cells to reduce the tetrazolium salt XTT to
orange-colored compound of formazon. The formed dye is water soluble and the
dye optical intensity can be determined at 490 nm. The intensity of the dye is
proportional to the number of metabolically active cells. The test procedure
includes cultivation of cells in 96-well plates, addition of the XTT reagent and
incubation for 2-24 hours, during which an orange color is formed. The greater
the number of metabolically active cells in the well, the greater the activity of
mitochondrial enzymes and the higher the concentration of the dye formed,
which can then be measured and quantified (117).
The Hela and PC-3 cells were seeded in 96-well plats at a density of 7000
cells/well, whereas the Hep3b cells were seeded at density 5000 cells/well. The
cells were maintained at (37C) for 24 hours in the presence of 100 l DMEM.
The medium was replaced with 8, 4, 2, 1, 0.5 or 0.25% plant extract-DMEM
concentration in triplicates. Twenty four hours later, 50 l of the XTT reaction
solution were added to each well and the plates were incubated at 37C for 3
hours. The absorbance was measured with ELISA reader at a wave length of
490 nm. The reference absorbance (non specific background reading) was
measured at 630nm. Negative control cells were incubated with no extract in the
medium .

27
The cells proliferative activity was estimated by calculating the ratio of remaining
viable cells in each well in comparison to the control and expressed as (% of
control). Each assay was repeated for tow additional times and the mean and
standard error was calculated for each extract concentration (113, 114, 115 and
116).
H. Trypan-blue dye exclusion Viability assay
The trypan-blue dye exclusion assay was used to determine the plant extract-
mediated cell death (89,110, 111 and 112). 200000 cells/well were seeded in 6-
well plates and grown as previously described. After 24 hours the medium was
replaced with different plant extract-DMEM concentration (8, 4, 2, 1, 0.5 and
0.25%) in triplicates. After 48 hours incubation the medium was discarded and
the cells were harvested by trypsinization and washed twice with (PBS). A
volume of 0.4% trypan-blue stain that is equal to the residual PBS was then
added. After 5 min incubation, the cells were counted with a hemocytometer by
compound light microscope. The unstained (viable) cells and the blue-stained
(dead) cells were counted separately.
Negative control cells were incubated with DMEM media without any extract and
treated the same way. Positive control cells were incubated for 10 min with
0.5mM H
2
O
2
before being harvested and counted as described.
The % cell viability was calculated using the following equation:
% cell viability = total viable cells (unstained) X 100 / total cells (stained + unstained)
Each experiment was carried out in triplicate and repeated for one more time and
the average of 6 wells was considered for each extract concentration.
I. Determination of morphological changes of the cells in culture
The different cell lines normally grown in 6-well plates or incubated with the
desired extract concentration for the purpose of viability testing were monitored
by an inverted microscope in 24 hours intervals. Any morphological changes in
28
the cells shape, level of adhesion and any other alterations were observed and
documented by photography before end of experiment (104, 114, 117 and 118).
J. Statistical analysis
All values expressed as mean standard error of the mean by Microsoft Excel.
The data were statistically analyzed by SPSS by performing the correlation,
regression and one-way ANOVA tests.
For each experiment the data obtained was blotted against extract concentration
and the obtained curve equation and R
2
value were calculated by the Microsoft
Excel software. The extract concentration that gives 50% or 100% reduction in
the number of metabolically active cells or in viability of cells (IC
50
and IC
100

respectively), was determined by substitution in the obtained equation (89.119).
29
0
100
200
300
400
500
600
700
800
900
1000
1100
0 24 48 72 96
Hours
C
e
l
l

n
u
m
b
e
r
1
0
0
0
Hep3b
Hela
PC3
RESULTS
I. Determination of the cell lines growth characteristics
Figure 5 shows the growth curve of each of the hepatocellular carcinoma Hep3B,
cervical epithelial Hela and Prostate PC-3 cell lines in normal DMEM culture
media. The three cell lines maintained exponential growth characteristics until the
end of experiments (96 hours). The Hep3b cells grew faster than the other two
cell lines. However, no cell line growth curve reached a plateau at the end of the
experiments (four days). It should be emphasized that the time of all of the
following experiments did not exceeded this time.

Figure 5. Growth curves of Hep3b, Hela and PC-3 cell lines.
Hepatocellular carcinoma Hep3b, Cervical epithelial Hela and
Prostate PC-3 Cells were seeded at a density of 100000 cells/well.
The wells were prepared in triplicates for each time point and were
incubated at 37C in 5% CO2. Three wells from each cell line were
harvested and counted at each time point and the rest of wells
allowed continuing growing. The numbers of cells were blotted
against the growth duration.
30
II. Determination of the different plant extracts working
concentrations
Plant extracts-media were prepared by introducing sterile plant extracts into the
DMEM culture media (the plant extract volume was included in final volume
calculation). The plant extract sterilization by a 0.2l filter was difficult to perform
as the filter was blocked by the fine pieces of extract. Thus the highest plant
concentration achieved (stock concentration) was 20% (wt/v). The examined
extract concentrations were 0, 4, 8 and 16% for each plant type. The effect of
such concentrations from Urtica pilulifera, Lagenaria siceraria and Ficus carica
on the viability of Hep3b cells is illustrated in Figure 6. Urtica pilulifera
concentrations less than 4 % (wt/v) showed a profound effect on the viability of
cells, while 4% and higher concentrations gave maximal effect of 100% cell
mortality. According to these result the gap between 0% and 4% is critical and a
wider range of concentrations in this interval are necessary. Lagenaria siceraria
gave weak reduction of Hep3b cells viability and the testing concentrations did
not give more than 70% effect. Ficus carica had lower but considerable effect on
the viability of Hep3b cells, and the highest concentrations did not reach the 50%
inhibition.
From Figure 6 it!s noticed that concentrations lower than 4% are needed to be
introduced into the experiment to assess cell death before reaching maximal
values. Moreover, it seems futile to examine concentrations higher than 8% as all
of the extract effects reach plateaus at those concentrations. Therefore the
concentrations (0.25, 0.5, 1, 2, 4 and 8%) of each plant extract were used to be
examined in the rest of the study. Any effect within this range of concentrations
would be amplified theoretically if we increased the concentrations.
31
0
10
20
30
40
50
60
70
80
90
100
110
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
Extracts concentrations.(dry wt / v)

P
e
r
c
e
n
t

v
i
a
b
i
l
i
t
y
Urtica pilulifera
Lagenaria siceraria
Ficus carica

Figure 6. A preliminary experiment of the effect of Urtica pilulifera,
Lagenaria siceraria and Ficus carica on the viability of
Hepatocellular carcinoma Hep3b cells. The percent viability of cells
was calculated as stated previously and plotted against the plants
concentrations.
32
III. Effect of plant extracts on cancer cells proliferation activity in
culture
A. Hepatocellular carcinoma cell line Hep3b
The Human hepatocellular carcinoma cell line Hep3b was maintained in the
presence of increasing concentrations of each of the three plant extracts as
indicated in each experiment. The following sections describe the effect of each
plant extract on this cell line in terms of proliferation activity (Figure 7).
1. Effect of Urtica pilulifera
Extracts from the plant Urtica Pilulifera were able to inhibit the proliferation of the
Hep3b cells in a dose response manner. The proliferation activity of the cells was
found to be significantly, inversely related to increasing the extract concentration
in the medium (P =0.013, R =-0.86). This inhibitory effect was initiated at low
extract levels and reached a 50% proliferation inhibition (IC
50
) when the cells
were grown in media with 1.9% extract concentration (R
2
=0.83). The highest
proliferation inhibition level was about 85% of the control group, however was
obtained at the maximum extract concentration tested (4%).
2. Effect of Lagenaria siceraria
A dose response effect was seen when the Hep3B cells were incubated with
increasing concentrations of the Lagenaria Siceraria extracts in the culture
medium. The degree of proliferation is also inversely related to the increase in
medium extract content. This relationship is significant with P-values of less than
or equal to 0.01and R values equal to -0.98. However, the extract inhibitory effect
was initiated at higher concentrations than in the case of Urtica Pilulifera. Higher
concentrations were thus needed to reach a 50% proliferation inhibition
(IC
50
=3.4%, R
2
= 0.97). The maximum extract concentration tested (8%) was able
to reach proliferation inhibition levels as high as in the case of Urtica pilulifera
(>85% of the control group).

33
3. Effect of Ficus carica
The results indicate that the proliferation of the Hep3B cells is inversely related to
the increased levels of Ficus carica extracts in there culture media. This behavior
is significant with P-values of >0.005 and R =-0.908. The extract concentrations
needed to elicit a considerable inhibitory effect were higher than the Urtica
pilulifera and Lagenaria Siceraria extracts (IC
50
=5.7%, R2=0.87). The maximal
inhibition observed (60% of the control group) was seen in media with 8% extract
content.
34
0
10
20
30
40
50
60
70
80
90
100
110
120
0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 5.5 6 6.5 7 7.5 8 8.5
Extracts concentrations (dry wt / v )
P
r
o
l
i
f
e
r
a
t
i
o
n

a
c
t
i
v
i
t
y

(

%

o
f

c
o
n
t
r
o
l

)
Lagenaria siceraria
Urtica pilulifera
Ficus carica

Figure 7. Reduction of proliferation activity of Hep3b cells in
response to plant extracts from Urtica pilulifera, Lagenaria siceraria
and Ficus carica.
5000 Hep3b cells/well were seeded in 96-well plates. The cells
were grown in DMEM culture media containing 8, 4, 2, 1, 0.5 or
0.25% of the indicated plant extract in triplicates. The cells!
proliferation activity was determined by the tetrazolium salt XTT
assay as described in the material and methods. Each experiment
was repeated for additional two times and the average of a total of
9 wells was calculated for each concentration. The results were
calculated as percent of the control group with no plant extract and
blotted against the respective extract concentration.
35
B. Cervical epithelial cell- line Hela
The cervical epithelial cell line Hela was grown in the presence of increasing
concentrations of plant extracts from Urtica pilulifera, Lagenaria siceraria and
Ficus carica. The following sections depict the effects of each plant extract on
Hela cells proliferation activity (Figure 8).
Urtica pilulifera extract showed a steep inhibitory effect on the proliferation of
Hela cells (figure 8). The proliferation activity of the cells was found to be inversly
related to the increase in extract concentration. A dose rsponse effect was
initiated at the lowest extract level used in the experiments (0.25%), and a 50%
inhibition of the cells proliferation activity was achevied at extract level of
(0.132%).
The proliferation activity of Hela cells was inhibited in a dose response manner
by Lagenaria siceraria. The proliferation activity of cells was found to be
significantly, inversely related to the increase in extract concentrations (P<0.01,
R=-0.938). The inhibitory effect of this extract was also started at the lowest
extract concentration used (0.25%) however higher concentrations were needed
(3.1%) to reach a 50% inhibition, than in the case of urtica pliulifera. Higher
concentration also were needed to reach the maximum levels of inhibition than in
the case of urtica pilulifera.
The results indicate that Ficus carica extract is also capable of reducing the Hela
cells proliferation in a concentration dependent manner. The proliferation activity
is also inversely related to the increase in extract concentration. This relationship
is significant with P > 0.05 and R= to -0.87. Although a considerable inhibitory
effect was induced by lower concentrations, IC
50
was reached at 3.1% extract
concentration. The effect of Ficus carica is slightly different from that of
Lagenaria siceraria, but with minor differences.
36
0
10
20
30
40
50
60
70
80
90
100
110
0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 5.5 6 6.5 7 7.5 8 8.5
Extracts concentrations ( dry wt /v)
P
r
o
l
i
f
e
r
a
t
i
o
n

a
c
t
i
v
i
t
y

(

%

o
f

c
o
n
t
r
o
l

)
Lagenaria siceraria
Ficus carica
Urtica piluifera

Figure 8. Reduction of proliferation activity of Hela cells in response
to plant extracts from Urtica pilulifera, Lagenaria siceraria and Ficus
carica.
7000 Hela cells/well were seeded in 96-well plates. The experiment
conditions were as in figure 7.
37
C. Prostate cell line PC-3
The prostate cells PC-3 were incubated in the presence of plant extracts from
Urtica pilulifera, Lagenaria siceraria of different concentrations. The inhibition of
proliferation activity of PC-3 is illustrated in the following sections (Figure 9).
Urtica pilulifera extract showed a strong inhibition of cell proliferation in a dose
response manner. The proliferation activity is inversely related to the increase in
extract concentration. This relationship is significant with P=0.004 and R= -0.916.
The 50% proliferation inhibition (IC
50
) was achieved at 2.3% extract content in the
medium. 4% extract concentration was able to reduce the proliferation activity to
reach about 10% only.
The results indicate that the proliferation activity of PC-3 cells is inversely related
to the increased levels of Lagenaria siceraria extracts concentration. Although
this proliferation inhibition was weaker than Urtica pilulifera effect, a significant
dose response manner was shown (P=0.00, R=-0.995). A relatively high extract
concentration 7.9% was necessary to reach 50% proliferation inhibition.
Ficus carica extract showed a significant inhibition of PC-3 proliferation activity
P= 0.001, R= -0.954. The lowest concentrations of this extract were able to
induce inhibitory effects more than the other extracts; despite of this noticeable
effect, IC
50
was 5.5% extract concentration.
38
0
10
20
30
40
50
60
70
80
90
100
110
0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 5.5 6 6.5 7 7.5 8 8.5
Extratcs concentrations ( dry wt /v )
P
r
o
l
i
f
e
r
a
t
i
o
n

a
c
t
i
v
i
t
y

(

%

o
f

c
o
n
t
r
o
l

)

lagenaria siceraria
Urtica pilulifera
Ficus carica

Figure 9. Reduction of proliferation activity of PC-3 cells in
response to plant extracts from Urtica pilulifera, Lagenaria siceraria
and Ficus carica.
7000 PC-3 cells/well were seeded in 96-well plates. The
experiment conditions were as in figure 7.
39
IV. Effect of plant extracts on cancer cell lines viability in culture
A. Hepatocellular carcinoma cell line Hep3B
Hep3b cells viability was determined in the presence of increasing concentrations
of plant extracts from Urtica pilulifera, Lagenaria siceraria and Ficus carica.
Figure 10, depicts the percent viability of Hep3b cells after incubation for 48
hours with different extracts at different concentrations. Hep3b cells which were
maintained in DMEM without any plant extract have a percent viability ranging
from 92 to 94.5% according to the three experiments.
The percent viability of Hep3b cells was found to be significantly, inversely
related to the increase in Urtica pilulifera extract concentration (P=0.03, R=-
0.787). The lowest concentration 0.25% of this extract was capable to reduce the
percent viability of the cells by slightly less than 30%. The 50% viability reduction
was obtained by 2.1% plant extract concentration. At 4% extract concentration
100% reduction in cell viability was already obtained.
The percent viability of Hep3b cells was reduced in a significant, dose response
manner due to treatment with Lagenaria siceraria (P=0.002, R=-0.937). Although
there is no noticeable inhibitory effect at the lowest concentration, the highest
concentration examined (8%) was able to elicit viability reduction by 60%. In
contrast with Urtica pilulifera behavior Lagenaria siceraria effect is weaker, and
IC
50
was obtained at 7.1% extract concentration.
The results show that increasing the levels of Ficus carica in the DMEM media is
inversely related to the percent viability of Hep3b cells. This behavior is
significant with P= 0.001 and R=-0.94. Despite of that both Ficus carica and
Lagenaria siceraria behave similarly at most of the concentrations examined,
higher Ficus extract concentration (18.1%) are estimated to be needed for
40
reaching the 50% reduction in cell viability, than Lagenaria siceraria extract
(6.7%). The difference between both extracts arrows at high concentration (8%).
0
10
20
30
40
50
60
70
80
90
100
110
0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 5.5 6 6.5 7 7.5 8 8.5
Extracts concentrations(dry wt /v)
P
e
r
c
e
n
t

v
i
a
b
i
l
i
t
y

(
%
o
f

c
o
n
t
r
o
l
)
Urtica pilulifera
Lagenaria siceraria
Ficus carica

Figure 10. Reduction of Hep3b cells viability in response to extracts
from Urtica pilulifera, Lagenaria siceraria and Ficus carica.
20000 Hep3b cells/well were seeded in 6-well plates. The cells
were grown in DMEM culture media containing 8, 4, 2, 1, 0.5 or
0.25% of the indicated plant extract in triplicates. The percent
viability was determined by the Trypan-blue test as described in the
materials and methods. Each experiment was repeated for one
additional time and the average Standard error of the mean, of a
total of 6-wells was calculated for each concentration. The results
were calculated as percent of the control group with no plant extract
and blotted against the respective extract concentration.
41
B. Cervical epithelial cells Hela
The percent viability of Hela cells was determined in the presence of increasing
concentrations of Urtica pilulifera, Lagenaria siceraria and Ficus carica plant
extracts. Figure11 shows the effect of each plant extract on this cell line in terms
of percent viability. In normal conditions without any extract treatment the percent
viability ranges from 90 to 95% according to the results of the three experiments.
Urtica pilulifera extract shows a steep and strong reduction of cell viability in a
dose response manner. This effect is significant with P=0.004, R=-0.911. This
effect was noticeable at 1% extract concentration, whereas to reach 50% viability
reduction, 2.8% extract concentration was required. A complete elimination of
viable cells was obtained at 4% extract concentration, and maintained at higher
concentrations.
The results illustrated in Figure11 indicate that Lagenaria siceraria extract causes
a significant viability reduction with P=0.00 and R=-0.982. This inhibitory effect is
weaker than the effect of Urtica pilulifera with IC
50
equals to 14.1% Lagenaria
siceraria extract concentration.
Ficus carica behaved similar to Lagenaria siceraria extract in causing some
reduction in percent viability of Hela cells. A dose response effect was observed
and significant with P= 0.003, R=-0.921. The fifty percent inhibition of cell viability
was at 15.7%. No cell viability reduction was achieved as much as in the case of
Urtica pilulifera even with the concentration 8% of both Lagenaria siceraria and
Ficus carica.
42
0
10
20
30
40
50
60
70
80
90
100
110
120
0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 5.5 6 6.5 7 7.5 8 8.5
Extarcts concentrations (dry wt /v)
P
e
r
c
e
n
t

v
i
a
b
i
l
i
t
y

(
%

o
f

c
o
n
t
r
o
l
)
Urtica pilulifera
Lagenaria siceraria
Ficus carica

Figure11. Reduction of Hela cells viability in response to extracts
from Urtica pilulifera, Lagenaria siceraria and Ficus carica.
The experiments! condition as in figure 10.
43
C. Prostate cell line PC-3
Figure 12 shows the percent viability of PC-3 cells grown in the presence of
increasing concentrations of plant extracts from Urtica pilulifera, Lagenaria
siceraria and Ficus carica. The following sections describe the effect of each
plant extract on this cell line in terms of percent viability. In normal conditions
without any extract addition, PC-3 cells showed a lower percent viability than
Hep3b and Hela cells. According to the three different experiments the percent
viability of PC-3 at normal DMEM was ranging between 85.5 and 90.5%.
Percent viability of PC-3 affected by Urtica pilulifera extract was significantly
inhibited in a dose response manner (P= 0.00 and R= -0.992). A smooth and a
steep relationship was initiated at the lowest extract concentration. The 50%
viability was obtained by 3.6% extract concentration, while 8% extract
concentration resulted in 100% elimination of any viable cells.
The percent cell viability was found to be significantly, inversely related to the
increase in extract concentrations (P= 0.001, R= -0.955). Despite of that this
effect is weaker than the previous extract effect, IC
50
of Lagenaria siceraria was
achieved at 9.4% extract concentration.
A dose response effect was also seen when PC-3 was grown in increasing
concentrations of Ficus carica. Percent viability was significantly, inversely
related to the increasing concentrations of this extract (P=0.00, R =-0.974).
Higher concentrations were needed to reach a 50% viability inhibition (IC
50

=13.8% extract concentration).
44
0
10
20
30
40
50
60
70
80
90
100
110
120
0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 5.5 6 6.5 7 7.5 8 8.5
Extracts concentrations (dry wt /v)
P
e
r
c
e
n
t

v
i
a
b
i
l
i
t
y
(
%

o
f

c
o
n
t
r
o
l
)
Urtica pilulifera
Lagenaria siceraria
Ficus carica

Figure 12. Reduction of PC-3 cells viability in response to extracts
from Urtica pilulifera, Lagenaria siceraria and Ficus carica
The experiment conditions where as in figure 10.
45
V. Determination of morphological changes of cell lines in
response to treatment with different plant extracts
The morphological characteristics of the Hep3b, Hela and PC-3 cell lines were
evaluated in parallel to each time performing viability testing assay. In each
viability testing experiment the 6-well plates were examined for any
morphological changes in response to treatment with the three plant extracts
(Urtica pilulifera, Lagenaria siceraria and Ficus carica) compared to the normally
maintained cells (no treatment). Representative wells were pictured and shown in
this study (Figures 13, 14 and 15). A few cell culture flasks were frequently
monitored at short time intervals.
A. Hepatocellular carcinoma cell line Hep3B
The normally maintained Hep3b cells were proliferating with high rate, and
formed a monolayer growth with no less than 90% confluency within 48 hours
(Figure 1a).
Urtica pilulifera showed noticeable morphological alterations with weaker
adhesion level at different concentrations as evident by the cells floating. After
48 hours, the 2% extract concentration showed a potent activity. It induced
Hep3b cells to round and form a grape-shaped cluster. At 24 hours after
treatment of hep3b with 4 and 8% extract most of cells were detached and
aggregated in clusters (Figure 16b)
Hep3b cells treated with Lagenaria siceraria 4 and 8% concentrations for 24
hours suffered from noticeable alterations such as shrinkage, sparse, and spindle
shape formation (Figure16c). At the highest concentration (8%) the level of
adhesion was reduced and many cells were detached.
46
Ficus carica treated cells showed a high proliferation rat at low extract
concentrations (0.25 and 0.5%). While at 4% extract concentration their growth
was inhibited and slight cell shrinkage was observed (figure 16d). On the other
hand 8% Ficus carica extract concentration induced obvious alterations to the
cells morphology such as, the rounding up and detachment of many cells.
a b
c d

Figure 13. Morphology of Hep3b cells in culture with or without
different plant extracts.
Hep3b cells were grown in DMEM culture medium without any
extract (a) or with 4% Urtica pilulifera extract for 24 hours (b), 8%
Lagenaria siceraria extract for 48 hours (c) and 4% Ficus carica for
48 hours (d). Cells in a, c and d were visualized by inverted
microscope at 10X and in b at 20X.
47
B. Cervical epithelial cells Hela
The proliferation rate of Hela cells maintained in normal media was lower than
Hep3b, as about 80% confluency was obtained after 48 hours of growth (Figure
17a). Apparent alterations in cell morphology and detachment of cells from the
culture surface were observed after 24 and 48 hour of treatment with different
extract concentrations.
Five to ten hours after treatment of Hela cells with Urtica pilulifera (2, 4 and 8%
concentrations) the cells began to round up and the level of adhesion was
influenced. Many cells detached easily from the surface of the plastic flasks and
moved into medium. After 24 hours of treatment with 2% Urtica pilulifera, most of
cells were rounded and formed sparse clusters (Figure 17b). Higher
concentrations (4 and 8%) had more pronounced effects and nearly all cells were
rounded up and detached.
When Hela cells were treated with 2% Lagenaria siceraria extract for 24 hours a
number of cells were dead as evident by cellular rounding up, floating and
fragmentations. The time and concentration dependent extract effect was
observed in the degree of confluency. Figure 7c illustrates the effect of 8%
extract concentration after 48 hours of incubation.
Treatment of Hela cells with Ficus carica 4 and 8% for 48 hours resulted in
reduced density of the monolayer, and some of the cells were round and
detached (Figure 17d).
48
a
d c
b

Figure 14. Morphology of Hela cells in culture with or without
Hela cells were grown in DMEM culture medium without any extract
(a) or with 2% Urtica pilulifera extract for 24 hours (b), 8%
48 hours (d). Cells in all were visualized by inverted microscope at
20X.
49
C. Prostate cell line PC-3:
Figure 18a shows PC-3 cells which are characterized by a considerably lower
proliferation rate than both Hela and Hep3b cell lines in normal conditions.
After 24 hours of PC-3 treatment with 4% Urtica pilulifera the cells were rounded
up, and detached from the monolayer (Figure 8b). There were relatively large
amounts of cellular fragments and cytoplasm condensation that appeared at 4%
Urtica pilulifera concentration.
PC-3 cells treated with 4 and 8% Lagenaria siceraria showed a noticeable
decrease in confluency after 48 hours of treatment, but no evident morphological
changes (Figure 18c).
The reduction of confluency degree was visible in PC-3 cells when incubated for
48 hours in the highest extract concentration used, but also with no noticeable
morphological changes (Figure 18d).
50
a
b
d c

Figure 15. Morphology of PC-3 cells in culture with or without
PC-3 cells were grown in DMEM culture medium without any
extract (a) or with 4% Urtica pilulifera extract for 24 hours (b), 8%
48 hours (d). Cells in all were visualized by inverted microscope at
20X.
51
DISCUSSION
Cancer is a term describing conditions characterized by unscheduled and
uncontrolled cellular proliferation (120). It is a very common disease, and its
incidence is increasing at an average annual rate of 1.2% (120). Lately, there has
been improvement in the treatment strategies of cancer, which has resulted in
prolonged survival of patients with chronic cancer disease. However there is a
growing need for additional means of cancer therapy, in the form of both
palliative and curative treatments. The strategies available today, are
sophisticated, and are only able to affect 50 to 60% of cancer patients, while the
others will eventually die from the disease (121, 122 and 123).
Chemotherapy has been used for cancer treatment for more than 50 years;
sometimes in combination with or parallel to surgery and radiotherapy. After
surgical ablation of progressive cancer, metastasized tumor cells continue to
progress and this is one of the faultiest associated with surgery. One the other
hand, radioactive rays and most anticancer chemotherapeutic agents damage
DNA or suppress DNA duplication to kill the rapidly growing tumor cells. At the
same time, they also affect normal cells causing serious adverse effects, such as
bone marrow function inhibition, bone necrosis, lung fibrosis, skin
devascularization, ulceration, nausea, vomiting, renal damage and alopecia
(124). Thus it is evident that a wide array of selective and potent components is
needed to match the growing problems associated with cancer.
Plants and natural products play an important role in medicine and provide
important prototypes for the development of novel drugs (125). They offer a
valuable source of compounds with a wide variety of biological activities and
chemical structures. Many anti cancer agents have been derived from natural
sources; directly as pure native compounds, or as semi-synthetic analogues
(126, 127 and 128).
52
Arabic and Islamic tradition is particularly rich in medical plants that have been
used by pioneer Arabic physicians to establish the basis for modern therapies.
But a few of these plants have been examined scientifically. In this extent we
studied the potential effect of three Arabic and Islamic traditional plants as
anticancer agents. These were Fig (Ficus carica) and Bottle gourd (Lagenaria
siceraria) that were mentioned in the holy Quran in more than one occasion and
Roman nettle (Urtica pilulifera) which is traditionally used as a popular
medication (8). To fulfill this aim, the proliferation, viability and morphological
characteristics of three cell lines (Hep3b, Hela and PC-3) were studied in
response to treatment with extracts from the aforementioned plants.
The cellular proliferation activity was tested by a colorimetric method which is
based on the ability of metabolically active cells to reduce the tetrazolium salt
XTT to orange-colored compounds of formazon. The intensity of the formed
water soluble dye is proportional to the number of metabolically active cells. The
proliferation activity of the treated cells was normalized to that of normally
growing cells from the same type with no treatment. This normalized value was
expressed as percent of the control group. Theoretically, any reduction in the
number of metabolically active proliferating cells might mean that the proliferation
pathway itself was halted (cell cycle arrest), or that a fraction of the cells went
through a death pathway. Therefore, a viability test was necessary to determine
which of these two options may play a role in this study. Therefore, the trypan-
blue dye exclusion test was used to determine the plant extract-mediated cell
death. The unstained cells (viable) and the blue stained (dead) were counted
separately, and the percent cell viability was calculated as previously described.
The viability assay was performed with the same cell-extract combinations as in
the proliferation assay. The percent viability of normally grown cells from the
same types was determined for comparison with extract-treated cells. If we
consider the results of the proliferation and viability assays in parallel, then we
would be able to say whether reduction of the number of the metabolically active
cell has resulted from death of part of them or due to any other reason. Any
conclusion drawn from such comparison may be confirmed morphologically
53
following up the treated cells in comparison with normally maintained control
cells. In this study a combination of the three different assays was performed in
parallel for each plant extract and each cell line.
In order to assess the degree of proliferation inhibition and viability reduction
obtained by each extract in comparison to the others, the IC
50
values were
summarized in tables 3 and 4. The findings of this study indicated that the
proliferation activity of Hep3b cells was inhibited by all of the three plant extracts
(Urtica pilulifera, Lagenaria siceraria and Ficus carica). A dose response effect
was obtained with all of these extracts, but the degree of this effect varied from
one extract to the other. The results indicated that Urtica pilulifera is the most
potent extract, even at low concentrations with IC
50
of (1.9%) followed by
Lagenaria siceraria with IC
50
of (3.4%) and Ficus carica with of IC
50
of (5.7%). At
the highest extract concentration used in the study (8%) both Urtica pilulifera and
Lagenaria siceraria reached a similar proliferation inhibition level of about (90%).
However this degree of inhibition was reached at lower concentration of Urtica
pilulifera extract than of Lagenaria siceraria extract (Figure7). Ficus carica on the
other hand did not reach the same degrees of proliferation inhibition obtained by
both Urtica pilulifera and Lagenaria siceraria even at the highest concentration.
When considering the results of Heb3b cells viability assay, the three plant
extracts showed various degrees of Heb3b cells viability reduction. Urtica
pilulifera showed a steep reduction of the cells viability with IC
50
of 2.1% extract
concentration, which equals the IC
50
of the same extract in cell proliferation
assay. These compatible results (Figure 16) suggest that reduction in the
metabolically active Hep3b cells in consequence to Urtica pilulifera extract
treatment is due to cell death. However such compatibility was not evident in the
case of Lagenaria siceraria (Figure 17) and Ficus carica (Figure 18). Lagenaria
siceraria has a less potent cytotoxic effect against Hep3b cells (IC
50
= 7.1%).
This might indicate that this extract counteracts the proliferation activity of Hep3b
cells mostly by mechanisms other than induction of their death. This deviation
between the cytotoxic effect and proliferation effect was more profound in the
54
case of Ficus carica with IC
50
of 18.1% in case of cytotoxicity compared to 5.7%
in the case of proliferation.
Table 3. Summary of calculated concentrations of the three plant
extracts Urtica pilulifera, Lagenaria siceraria and Ficus carica that give
50% (IC
50
) reduction in proliferation activity of Hep3b, Hela and PC-3
cancer cell lines. The R
2
Plant Hep3b cells Hela cells PC-3 cells
IC
50
1.98 0.132 2.31 Urtica pilulifera
R
2
0.837 0.6022 0.8563
IC
50
3.4 3.1 7.9 Lagenaria siceraria
R
2
0.97 0.9761 99.82
IC
50
5.7 3.1 5.5 Ficus carica
R
2
0.8675 0.9059 0.9583

Table 4. Summary of calculated concentrations of the three plant
extracts Urtica pilulifera, Lagenaria siceraria and Ficus carica that give
50% (IC
50
) reduction in the % viability of Hep3b, Hela and PC-3 cancer
cell lines. The R
2
Plant Hep3b cells Hela cells PC-3 cells
IC
50
2.1 2.8 3.6 Urtica pilulifera
R
2
0.7056 0.8305 0.9843
IC
50
7.16 14.18 9.4 Lagenaria siceraria
R
2
0.8496 0.9585 0.9367
IC
50
18.12 15.7 13.8 Ficus carica
R
2
0.8886 0.852 0.9442
55
R
2
= 0.837
-20
-10
0
10
20
30
40
50
60
70
80
90
100
110
0 0. 5 1 1.5 2 2. 5 3 3. 5 4 4.5 5 5.5 6 6. 5 7 7.5 8 8. 5
Extrac t concentrati ons ( dry wt / v)
P
e
r
c
e
n
t

o
f

e
f
f
e
c
t
prolif eration
of vi abili ty
R
2
= 0.6022
-30
-20
-10
0
10
20
30
40
50
60
70
80
90
100
110
0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 5.5 6 6.5 7 7.5 8 8.5
Extract concentrations (dry wt /v)
P
e
r
c
e
n
t

o
f

e
f
f
e
c
t

proliferation
viability
R
2
= 0. 85 63
-10
0
10
20
30
40
50
60
70
80
90
100
110
0 0.5 1 1.5 2 2. 5 3 3.5 4 4.5 5 5. 5 6 6.5 7 7. 5 8 8.5
Extr ac t c onc entrati ons( dry wt /v)
P
e
r
c
e
n
t

o
f

e
f
f
e
c
t
Pr oli ferati on
Vi abi li ty
Figure16 : Reduction of viability and proliferation
activity of Hep3b (a), Hela (b) and PC3 (c) cells in
response to Treatment with Urtica pilulifera extract.
The best-fit trend line are shown. The equation for each
trend l ine was used to estimate the IC50, IC100 and R2
values by substitution. R2 values Are indicated next to
the respective l ines.
a
b
c

56
R
2
= 0.8496
R
2
= 0.97
0
10
20
30
40
50
60
70
80
90
100
110
120
0 0.5 1 1. 5 2 2.5 3 3.5 4 4.5 5 5. 5 6 6.5 7 7. 5 8 8.5
Extract conc entrations (dry wt / v)
P
e
r
c
e
n
t

o
f

e
f
f
e
c
t

viability
proliferation
R
2
= 0. 9585
R
2
= 0. 9761
0
10
20
30
40
50
60
70
80
90
100
110
0 0.5 1 1.5 2 2.5 3 3.5 4 4. 5 5 5.5 6 6.5 7 7.5 8 8.5
Extr act c oncentrati ons(dr y wt /v)
P
e
r
c
e
n
t

o
f

e
f
f
e
c
t
Proli feration
viabi li ty
R
2
= 0.9367
R
2
= 0.9982
0
10
20
30
40
50
60
70
80
90
100
110
0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 5.5 6 6.5 7 7.5 8 8.5
P
e
r
c
e
n
t

o
f

e
f
f
e
c
t
proliferation
Viability
response to Treatment with Lagenaria siceraria
extract.
trend line was used to estimate the IC
50
, IC
100
and R
2
values by substitution. R
2
values Are indicated next to
the respective lines.
a b
c

57
R
2
= 0.9442
R
2
= 0.9583
0
10
20
30
40
50
60
70
80
90
100
110
0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 5.5 6 6.5 7 7.5 8 8.5
Extract concentrations (dry wt / v )
P
e
r
c
e
n
t

o
f

e
f
f
e
c
t
Proliferation
Viability
R
2
= 0.852
R
2
= 0.9059
0
10
20
30
40
50
60
70
80
90
100
110
0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 5.5 6 6.5 7 7.5 8 8.5
p
e
r
c
e
n
t

o
f

e
f
f
e
c
t
Proliferation
viabiltity
R
2
= 0.8886
R
2
= 0.8675
0
10
20
30
40
50
60
70
80
90
100
110
0 0.
5
1 1.
5
2 2.
5
3 3.
5
4 4.
5
5 5.
5
6 6.
5
7 7.
5
8 8.
5
Extract concentrations(dry wt / v)
p
e
c
e
n
t

o
f

e
f
f
e
c
t
viability
proliferation
response to Treatment with Ficus carica extract.
trend line was used to estimate the IC50, IC100 and R2
values by substitution. R2 values Are indicated next to the
respective lines.
a b
c
58
These data were confirmed by the inspection of morphological alterations of
Hep3b cells due to treatment with the three plant extracts (Figure13). As shown
in figure 13b Urtica pilulifera had the most potent effect compared to the control
(Figure 13a). Most of cells were rounded up and formed a grape shaped cluster,
which is one of the prominent morphological features of cellular death in culture.
Such features were also noticeable to lesser extent when the cells were
incubated with Lagenaria siceraria, and could hardly be detected in the case of
Ficus carica. Moreover, the confluency of Heb3b cells was reduced in response
to treatment with the three plant extracts and in accordance to the previously
discussed results of proliferation inhibition ( 104,114, 118).
Similarly Hela cells proliferative activity was inhibited with various levels due to
their treatment with the three plant extracts. As shown in Figure 8, Urtica
pilulifera has a strong inhibitory effect on the proliferation activity in extract
concentration dependent manner with IC
50
of 0.13% extract concentration. Both
Ficus carica and Lagenaria siceraria had a similar but weaker effect on
proliferation of Hela cells, and the IC
50
for both extracts was 3.1%. The percent
viability of Hela cells was also reduced in a dose response manner by all of these
plant extracts. Urtica pilulifera extract was the most effective with IC
50
of 2.8%,
while the Lagenaria siceraria and Ficus carica effect on cells viability were
similarly less potent with IC
50
of 15.7% for Ficus carica and 14.1% for Lagenaria
siceraria. Being the most potent plant, Urtica pilulifera mostly exerts its effects by
inducing cell death although other effect can be present (IC50 for
proliferation=0.13% and for cytotoxicity =2.8%). The difference between the IC
50
of both Ficus carica and Lagenaria siceraria in proliferation inhibition and the IC
50

of cell viability reduction indicates that these extracts exert their effect by cell
cycle arrest with low cytotoxicity. The morphological observations of Hela cells
maintained in increasing concentrations of the three plant extracts show that
Urtica pilulifera had the most rapid and potent effect of these extracts. Figure 14b
shows that most of the cells were rounded up and formed sparse clusters due to
treatment with 2% Urtica pilulifera. These results suggest that most of Hela cells
were dead in the case of Urtica pilulifera. A reduction of Hela cells confluency
59
was the most prominent observation when these cells were incubated with Ficus
carica or Lagenaria siceraria, but cell death indications were almost not seen.
These observations indicate an antiproliferative effect of both Ficus carica and
Lagenaria siceraria but less likely cytotoxicity.
The proliferation activity of the prostate cell line PC-3 was inversely related to
increasing the levels of the three plant extracts. The degree of proliferation
reduction varied from one plant extract to the other. As shown in figure 9, Urtica
pilulifera was again the most potent extract with IC
50
of 2.3%, whereas Ficus
carica comes second with IC
50
of 5.5%, and Lagenaria siceraria comes last with
IC
50
of 7.9%. When comparing these results with results from PC-3 cells viability
assay, we find that Urtica pilulifera also has the strongest effect with IC
50
of 3.6%.
These results are supported by the morphological changes shown in figure15b
including cells rounding up and detachment from the monolayer indicating that
cell death might has occurred.
Lagenaria siceraria induced a reduction of PC-3 cell viability with IC
50
of 9.4%
extract concentration similar to IC
50
from the same extract in the proliferation
assay. This fact suggests that this extract also caused PC-3 cell death. The
reduction of the cells confluency due to Lagenaria siceraria treatment observed
in figure 14c gives evidence of the effect of this extract. A Higher concentration of
Ficus carica was needed to reach a 50% viability inhibition (IC
50
=13.8%) than
that needed to reach 50% inhibition in cell proliferation (IC
50
=5.5%). This again
suggests that the effect of this extract did not occur only through cell death.
Based on the previous results, Urtica pilulifera have a potent antiproliferative
effect on the three tested cancer cell lines. While there is no previous studies
about the role of Urtica pilulifera extract as anticancer agent, many studies
investigated the role of its genus member Urtica dioica. Urtica pilulifera and
Urtica dioica are similar in many chemical and morphological aspects (75, 8). Our
results on Urtica pilulifera are in agreement with previous studies on Urtica
dioica. For example an aqueous extract of Urtica dioica roots was shown to
60
directly inhibit the proliferation of Hela cells and block its binding by epidermal
growth factor (EGF) (129). In the same study a polysaccharide mixture from an
aqueous root extract was shown to exert an anti-inflammatory activity in a rat
Paw oedema test. Investigation of the effect of a 20% methanol extract of Urtica
dioica roots on prostate cell line (LNCap) resulted in a selective and significant
concentration-dependent proliferation reducing effect on prostate cells (89). In
the same study the cell proliferation activity was determined by a colorimetric
assay and the cytotoxicity was examined by Trypan-blue test. Our results are
also comparable with data obtained from an in vitro study that aimed to
investigate the effect of Urtica dioica leaves aqueous extract on the enzyme
activity of prostate cancer tissue (130). The results of the study indicated that the
extract caused a significant inhibition of adenosine deaminase activity (ADA) of
these tissues in a dose dependent manner. These data might be of importance
because ADA is a key enzyme in the nucleotide metabolism and DNA turn over.
Extracts of Urtica dioica were also used in the treatment of adult mouse with
bingeing prostate hyperplasia (131). Five differently prepared root extracts were
tested on these rats. The 20% methanol extract was the most effective with
51.4% inhibition of induced growth.
Despite all of these data the mechanism(s) of action of Urtica dioica extract as an
antiproliferative agent has yet to be established. Different modes of actions are
proposed in this regard. For example it has been observed that some sterols and
hydroxyl-fatty acids, given their low concentrations in Urtica dioica, can inhibit
aromatase, which is a key enzyme in steroid hormone metabolism mediating the
conversion of androgens into estrogens (132). Another mechanism involves a
dose dependent inhibition of the binding of sex hormone binding gluobin (SHBG)
to its receptor in response to Urtica dioica extract (133). Some lignans present in
Urtica dioica were shown to interfere with the binding of androgens to SHBG,
thereby reducing the transport capacity of androgens (132). Based on the
similarity between Urtica pilulifera and Urtica dioica we would suggest that Urtica
pilulifera might exerts its antiproliferative effect via a similar fashion.
61
The results of this study strongly indicate a possible cytotoxic effect of Urtica
pilulifera against cancer cell lines. This was evident from the results of Trypan-
blue viability assay as well as from the inspection of cells morphology in culture.
The cytotoxicity of fixed and volatile oils extracted from Urtica pilulifera leaves
and seeds were tested on Swiss albino mice (134). The results indicated that
both oils of Urtica pilulifera are completely nontoxic even at doses reaching 12.8
ml/kg. However this study involved only the oil fractions of Urtica pilulifera
extract. Other components of the extract may play a role in the observed toxicity
in our study. Moreover, the aforementioned study was performed in vivo on mice
and the only cytotoxicity parameter considered was the mice death. No other
mice toxicity indicators were analyzed such as, histopathological alterations of
the different mice tissues and organs.
According to results discussed earlier the inhibitory role of Ficus carica on the
proliferation of the three cell lines was more profound than its cytotoxic effect on
the same cell lines. This conclusion is in accordance with the results of previous
studies. For instance, the proliferation of different cell lines was inhibited by
components of Ficus carica (135). Such proliferation inhibition was also obtained
when cow teat papillomatosis skin surface benign tumors, were treated by Ficus
carica latex (136). Furthermore, Ficus carica was found to have an in vivo
antioxidant effect after being consumed by human (137). Accordingly, the dried
fruits of this plant should be eaten more frequently as they are rich in phenol
antioxidants and fibers. Compared with vitamins C and E, the well known
antioxidants, the dried fruits of Ficus carica were found to be a superior one.
Lagenaria siceraria was found to posses both antiproliferative effects as well as a
cytotoxic effect to a considerable degree. Unfortunately no literature was found
regarding the anticancer activities of this plant. However, members of its family
(Cucurbitaceae), were shown to have a class of biologically active compounds
(Cucurbitacins) with well established anticancer cytotoxic activity (138,139,140).
For example, Cucurbitacins were shown to have strong cytotoxic effect on KB
cell line, derived from human nasopharyngeal carcinoma and on Hela cells by
62
different proposed mechanisms (140, 141). Moreover, (Cucurbitacins) were
shown to have a proliferative inhibitory activity in vivo against several carcinoma,
sarcoma, and leukemia models (142, 143 and 144).
The level of these compounds and/or any of their derivatives in Lagenaria
siceraria is unknown. However the results of this thesis indicate both
antiproliferative and cytotoxic activity of Lagenaria siceraria. Therefore we expect
Lagenaria siceraria extracts to behave in a similar manner like other members of
its family via (Cucurbitacins) involvement.
63
CONCLUSION
In conclusion, all of the three plants examined in this study possess varying
levels of anticancer activity in vitro. This is evident by the concentration
dependent manner reduction in the final number of cancer cells as a
consequence to treatment. Two kinds of anticancer effects were examined and
found to take part in this study. The first is anti-cell proliferation effect (decreased
number of metabolically active cells) and the second is cytotoxicity (decreased
number of live cells). The three plants examined possess both of the effects with
various degrees. Urtica pilulifera possess the strongest and most profound
effects on the three cell lines, probably by cytotoxicity mainly. On the other hand
Lagenaria siceraria probably affects the three cell lines by combination of
cytotoxicity and antiproliferation almost to a similar degree. Ficus carica most
probably reduces the final number of metabolically active cells mainly by its
antiproliferative effect, although cytotoxicity likely contributes to viability reduction
at high concentrations.
Both Ficus carica and Lagenaria siceraria are edible plants that were chosen on
the bases of being mentioned in the holy Quran. Therefore, although their effect
is lower than that of Urtica pilulifera their amount in the diet or as a treatment can
be safely scaled up when ingested in their native form. On the other hand,
despite its possible toxicity Urtica pilulifera is frequently orally used as a
medication in many conditions by traditional medicine.
64
RECOMMENDATIONS
1. Further studies are needed to assess the active ingredients of Ficus
carica, Lagenaria siceraria and Urtica pilulifera, involved in the
antiproliferative or cytotoxic effects of these plants. These studies must
involve the establishment of in vivo cancer models and their treatment by
these plants in their native crude extracts or by their purified active
components. More efficient extraction techniques and instruments have to
be used in order to obtain the crude extracts or their derivatives in
convenient quantities and concentrations.
2. The type(s) of effects exerted by the three plants component(s) involved in
their activity have to be further studied by molecular or other relevant
techniques.
3. Both Ficus carica and Lagenaria siceraria may be ingested more
frequently in different forms, if not to treat cancers, then as a prophylactic
against them. Urtica pilulifera can be administered for treating relevant
conditions including cancer with reasonable pre-studied amounts.
4. Many other plants are waiting to be examined for any possible therapeutic
effects and if present they may represent a convenient and cheap means
to cope with many types of cancer.

65
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